Substituted (e)-n&#39;-(1-phenylethylidene)benzohydrazide analogs as histone demethylase inhibitors

ABSTRACT

In one aspect, the invention relates to substituted (E)-N′-(1-phenylethylidene)benzohydrazide analogs, derivatives thereof, and related compounds, which are useful as inhibitors of lysine-specific histone demethylase, including LSD1; synthetic methods for making the compounds; pharmaceutical compositions comprising the compounds; and methods of using the compounds and compositions to treat disorders associated with dysfunction of the LSD1. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

BACKGROUND

Over the past decade it has become clear that epigenetic changes, whichalter gene activity without altering DNA sequence, collaborate withgenetic mistakes to promote cancer development and progression (Tsai, H.C. and Baylin, S. B. Cell Res 2011, 21 (3), 502-17; and Fullgrabe, J.,Kavanagh, E., and Joseph, B. Oncogene 2011). The regulation of themodifications on DNA and the proteins associated with DNA has become anarea of intense interest and the enzymes involved in these processeshave been suggested as a new class of protein targets for drugdevelopment. The major proteins associated with DNA are histoneproteins. Histone tails are subject to a variety of posttranslationalmodifications, such as phosphorylation, acetylation, methylation, andubiquitination, and these modifications, especially acetylation andmethylation on lysine residues, play a major role in the regulation ofgene expression, and are often dysregulated in cancer (Fullgrabe, J.,Kavanagh, E., and Joseph, B. Oncogene 2011).

Recently an enzyme called Lysine-Specific Demethylase 1 (LSD1) was foundto catalyze the oxidative demethylation of monomethylated anddimethylated histone H3 at lysine 4 (H3K4me1 and H3K4me2) and lysine 9(H3K9me1 and H3K9me2) through a flavin adenine dinucleotide(FAD)-dependent reaction (Shi, Y., et al. Cell 2004, 119 (7), 941-53;and Metzger, E., et al. Nature 2005, 437 (7057), 436-9), Whereas histoneacetylation is associated with loose chromatin and gene activation,methylation of histones is less straightforward. Using the lysineresidues regulated by LSD1 as an example, methylation at H3K4 isgenerally associated with gene activation, while methylation of H3K9 isassociated with transcriptional repression.

There is currently one known mammalian homolog of LSD1 which is aprotein variously designated LSD2, KDM1b, and AOF1. It shares a similardomain homology, but exhibits less than 31% sequence identity (Fang, R.et al. Molecular Cell 2010, 39:222-233). It has been shown that LSD2 isa H3K4me1/2 demethylase that specifically regulates histone H3K4methylation within intragenic regions of its target genes (ibid.). BothLSD1 and LSD2 contain a SWIRM domain, a FAD coenzyme-binding motif, anda C-terminal amine oxidase domain, all of which are critical to theenzymatic activity. However, unlike LSD1, the protein LSD2 contains aCW-type zinc finger domain in its N-terminal domain, a region which isunstructured in LSD1. Furthermore, LSD2 lacks the “tower domain” ofLSD1. At a cellular level, it has been suggested that LSD2 has a role intranscriptional regulation (ibid.). As expected, LSD2 appears to play arole in regulating DNA methylation as well, although the role in DNAmethylation may be developmental stage specific (ibid.; Ciccone, D. N.,et al. Nature 2009 461:415-418; Karytinos, A., et al. J. Biol. Chem.2009 284:17775-17782; and Yang, Z., et al. Cell Res. 2010 20:276-287).

Several lines of evidence point to LSD1 as being a possible therapeutictarget in cancer. LSD1 is reportedly over-expressed in a variety oftumors including neuroblastoma, ER-negative breast, bladder, lung, andcolorectal tumors (Schulte, J. H., et al. Cancer Res 2009, 69 (5),2065-71; Lim, S., et al. Carcinogenesis 2010, 31 (3), 512-20; andHayami, S., et al. Int J Cancer 2011, 128 (3), 574-86). Increasedmethylation of the permissive H3K4 mark by LSD1 inhibition has beenshown to reactivate expression of tumor suppressor genes in cancermodels (Huang, Y., et al. Clin Cancer Res 2009, 15 (23), 7217-28). Inaddition, LSD1 has been found to associate with estrogen and androgenreceptors leading to the specific demethylation of the repressive H3K9mark, thereby increasing target gene expression (Metzger, E., et al.Nature 2005, 437 (7057), 436-9; and Garcia-Bassets, I., et al. Cell2007, 128 (3), 505-18). Thus, depending upon cofactors bound to LSD1,demethylation by LSD1 can contribute to cancer through both thepermissive H3K4 and the repressive H3K9 mark. Therefore, the inhibitionof LSD1 might be an effective strategy for re-expression ofepigenetically silenced tumor suppressor genes as well as downregulation of important cancer pathways in a number of cancer types.Several LSD1 inhibitors have been reported, but they have shown poorselectivity and/or pharmacological properties, making furtherexploration of LSD1 biology difficult.

Monoamine oxidase (MAO) inhibitors such as tranylcypromine and pargylinehave been reported as LSD1 inhibitors, and there have been severalreports regarding attempts to discover derivatives with increasedselectivity for LSD1 over MAO (Mimasu, S., et al. Biochemistry 2010, 49(30), 6494-503; Binda, C., et al. J Am Chem Soc 2010, 132 (19), 6827-33;Culhane, J. C., et al. J Am Chem Sac 2006, 128 (14), 4536-7; Culhane, J.C., et al. J Am Chem Soc 2010, 132 (9), 3164-76; and Ueda, R., et al. JAm Chem Soc 2009, 131 (48), 17536-7). These compounds irreversiblyinactivate LSD1 by covalent binding to the FAD cofactor. Polyaminederivatives have also been evaluated as LSD1 inhibitors, where compoundswith activity in the μM range have been described (Huang, Y., et al.Clin Cancer Res 2009, 15 (23), 7217-28; Sharma, S. K., et al. J Med Chem2010, 53 (14), 5197-212; and Huang, Y., et al. Proc Nat/Acad Sci USA2007, 104 (19), 8023-8). In general, these and other reported LSD1inhibitors are neither adequately selective nor potent enough tooptimally interact with the crucial amino acid residues of thesubstrate-binding site present in LSD1.

In summary, the LSD proteins play a key role in epigenetic andtranscriptional regulation, and they are frequently altered in mammaliancancers, thus making them an attractive target for therapeuticintervention. Despite advances in drug discovery directed to identifyinginhibitors of LSD1 and/or LSD2 protein activity, there is still ascarcity of compounds that are both potent, efficacious, and selectiveinhibitors of either LSD1 or LSD2. Furthermore, there is a scarcity ofcompounds effective in the treatment of cancer and other diseasesassociated with dysfunction in LSD1 and/or LSD2. These needs and otherneeds are satisfied by the present invention.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates tocompounds useful as inhibitors of lysine-specific demethylase, or LSD.In a further aspect, the disclosed compounds and products of disclosedmethods of making, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof, are modulators of LSD activity, methodsof making same, pharmaceutical compositions comprising same, and methodsof treating disorders associated with a LSD activity dysfunction usingsame. In a still further aspect, the present invention relates tocompounds that bind to a LSD protein and negatively modulate LSDactivity. The disclosed compounds can, in one aspect, exhibit subtypeselectivity. In a further aspect, the disclosed compounds exhibitselectivity for the LSD1 member of the LSD protein family. In a stillfurther aspect, the disclosed compounds exhibit selectivity for the LSD2member of the LSD protein family.

Also disclosed are pharmaceutical compositions comprising, atherapeutically effective amount of a disclosed compound and apharmaceutically acceptable carrier.

Also disclosed are synthetic methods for making the disclosed compounds.In a further aspect, disclosed are the products of the disclosedsynthetic methods.

Disclosed are methods for the treatment of a disorder associated with aLSD activity dysfunction in a mammal comprising the step ofadministering to the mammal a therapeutically effective amount of adisclosed compound, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof.

Also disclosed are methods for inhibition of LSD activity in a mammalcomprising the step of administering to the mammal a therapeuticallyeffective amount of least one disclosed compound, or a pharmaceuticallyacceptable salt, hydrate, solvate, or polymorph thereof.

Also disclosed are methods for inhibiting LSD activity in at least onecell, comprising the step of contacting the at least one cell with aneffective amount of least one disclosed compound, or a pharmaceuticallyacceptable salt, hydrate, solvate, or polymorph thereof.

Also disclosed are uses of a disclosed compound, or a pharmaceuticallyacceptable salt, hydrate, solvate, or polymorph thereof. In a furtheraspect, the invention relates to pharmaceutical compositions comprisinga pharmaceutically acceptable carrier and an effective amount of adisclosed compound, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof.

Also disclosed are kits comprising at least one disclosed compound, or apharmaceutically acceptable salt, hydrate, solvate, or polymorphthereof, and one or more of: (a) at least one agent known to increasehistone demethylase activity; (b) at least one agent known to decreasehistone demethylase activity; (c) at least one agent known to treat adisorder of uncontrolled cellular proliferation; (d) at least one agentknown to treat a neurodegenerative disorder; (e) instructions fortreating a neurodegenerative disorder; or (f) instructions for treatinga disorder associated with uncontrolled cellular proliferation.

Also disclosed are methods for manufacturing a medicament comprising,combining at least one disclosed compound or at least one disclosedproduct with a pharmaceutically acceptable carrier or diluent. In afurther aspect, the invention relates to the use of a disclosed compoundin the manufacture of a medicament for the treatment of a disorderassociated with a LSD activity dysfunction. In a yet further aspect, theLSD activity dysfunction is a LSD1 activity dysfunction. In an evenfurther aspect, the LSD activity dysfunction is a LSD2 activitydysfunction. In a still further aspect, the invention relates to theused of disclosed compound in the manufacture of a medicament for thetreatment of a disorder of uncontrolled cellular proliferation.

Also disclosed are uses of a disclosed compound or a disclosed productin the manufacture of a medicament for the treatment of a disorderassociated with a LSD dysfunction in a mammal.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

A. DEFINITIONS

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asChemDraw™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the term “LSD” refers collectively to either or bothLSD1 and LSD2.

As used herein, the terms “LSD1” and “lysine-specific demethylase 1” canbe used interchangeably and refer to a histone demethylase encoded bythe KDM1A gene. The KDM1A gene has a gene map locus of 1p36.12 asdescribed by the Entrez Gene cytogenetic band, Ensembl cytogenetic band,and the HGNC cytogenetic band. The term LSD1 refers to a native proteinthat has 852 amino acids with a molecular weight of about 92903 Da, andis a member of the flavin monoamine oxidase family. The term LSD1 isinclusive of the protein, gene product and/or gene referred to by suchalternative designations as: LSD1, KDM1; RP1-184J9.1; AOF2; BHC110;KIAA0601; LSD1; BRAF35-HDAC complex protein BHC110; FAD-binding proteinBRAF35-HDAC complex, 110 kDa subunit; amine oxidase (flavin containing)domain 2; lysine-specific histone demethylase 1; lysine-specific histonedemethylase 1A; flavin-containing amine oxidase domain-containingprotein 2; lysine (K)-specific demethylase 1; amine oxidase (flavincontaining) domain 2; and FAD-binding protein BRAF35-HDAC complex, 110kDa subunit, as used by those skilled in the art.

As used herein, the terms “LSD2 and “lysine-specific demethylase 2 canbe used interchangeably and refer to a histone demethylase encoded bythe KDM1B gene. The KDM1B gene has a gene map locus of 6p22.3 asdescribed by the Entrez Gene cytogenetic band, Ensembl cytogenetic band,and the HGNC cytogenetic band. The term LSD21 refers to a native proteinthat has 822 amino acids with a molecular weight of about 92098 Da, andis a member of the flavin monoamine oxidase family. The term LSD2 isinclusive of the protein, gene product and/or gene referred to by suchalternative designations as: LSD2, AOF1; FLJ33898; FLJ34109; FLJ43328;C6orf193; DKFZp686I0412; OTTHUMP00000179125; bA204B7.3; dJ298J15.2;flavin-containing amine oxidase domain-containing protein 1;lysine-specific histone demethylase 2; lysine (K)-specific demethylase1B; amine oxidase (flavin containing) domain 1; amine oxidase, flavincontaining 1; lysine-specific histone demethylase 2; chromosome 6 openreading frame 193; and lysine-specific histone demethylase 1B, as usedby those skilled in the art.

As used herein, the term “histone demethylase” refers to that group ofenzymes which remove methyl groups from histone proteins. The term isinclusive of both histone lysine demethylases, i.e. enzymes which removemethyl groups from lysine residues in histones, and histone argininedemethylases, i.e. enzymes which remove methyl groups from arginineresidues in histones.

As used herein, the term “histone lysine demethylase” or“lysine-specific histone demethylase” can be used interchangeably, andboth refer to that group of enzymes which remove methyl groups fromlysine residues of histone proteins. The histone lysine demethylases area group of enzymes which comprise the following specific forms: LSD1,LSD2, JMJD2A, JMJD2B, JMJD2C and JMJD2D.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some aspects of the disclosedmethods, the subject has been diagnosed with a need for treatment of adisorder of uncontrolled cellular proliferation associated with ahistone lysine demethylase dysfunction prior to the administering step.In some aspects of the disclosed method, the subject has been diagnosedwith a need for inhibition of a histone lysine demethylase prior to theadministering step.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In various aspects, the term covers anytreatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In one aspect, the subject is a mammal such as a primate, and, in afurther aspect, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse,rabbit, rat, guinea pig, fruit fly, zebra fish etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with a disorder of uncontrolled cellular proliferation” meanshaving been subjected to a physical examination by a person of skill,for example, a physician, and found to have a condition that can bediagnosed or treated by a compound or composition that can inhibit ahistone lysine demethylase. As a further example, “diagnosed with a needfor inhibition of a histone demethylase” refers to having been subjectedto a physical examination by a person of skill, for example, aphysician, and found to have a condition characterized by a histonedemethylase dysfunction. Such a diagnosis can be in reference to adisorder, such as a disorder of uncontrolled cellular proliferation,cancer and the like, as discussed herein. For example, the term“diagnosed with a need for inhibition of histone demethylase activity”refers to having been subjected to a physical examination by a person ofskill, for example, a physician, and found to have a condition that canbe diagnosed or treated by inhibition of histone demethylase activity.For example, “diagnosed with a need for treatment of one or moredisorders of uncontrolled cellular proliferation associated with ahistone demethylase dysfunction” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have one or more disorders of uncontrolled cellularproliferation associated with a histone demethylase dysfunction.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a disorder relatedto a dysfunction of histone demethylase activity) based upon an earlierdiagnosis by a person of skill and thereafter subjected to treatment forthe disorder. It is contemplated that the identification can, in oneaspect, be performed by a person different from the person making thediagnosis. It is also contemplated, in a further aspect, that theadministration can be performed by one who subsequently performed theadministration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration,intraurethral administration, and parenteral administration, includinginjectable such as intravenous administration, intra-arterialadministration, intramuscular administration, and subcutaneousadministration. Administration can be continuous or intermittent. Invarious aspects, a preparation can be administered therapeutically; thatis, administered to treat an existing disease or condition. In furthervarious aspects, a preparation can be administered prophylactically;that is, administered for prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, cell, etc.), either directly; i.e., byinteracting with the target itself, or indirectly; i.e., by interactingwith another molecule, co-factor, factor, or protein on which theactivity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side affects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex, and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, “EC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50% agonismor activation of a biological process, or component of a process,including a protein, subunit, organelle, ribonucleoprotein, etc. In oneaspect, an EC₅₀ can refer to the concentration of a substance that isrequired for 50% agonism or activation in vivo, as further definedelsewhere herein. In a further aspect, EC₅₀ refers to the concentrationof agonist or activator that provokes a response halfway between thebaseline and maximum response.

As used herein, “IC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50%inhibition of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. For example, anIC₅₀ can refer to the concentration of a substance that is required for50% inhibition in vivo or the inhibition is measured in vitro, asfurther defined elsewhere herein. Alternatively, IC₅₀ refers to the halfmaximal (50%) inhibitory concentration (IC) of a substance Theinhibition can be measured in a cell-line such as AN3 CA, BT-20, BT-549,HCT 116, HER218, MCF7, MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468,PANC-1, PC-3, SK-N-MC, T-47D, and U-87 MG. In a yet further aspect, theinhibition is measured in a cell-line, e.g. HEK-293 or HeLa, transfectedwith a mutant or wild-type mammalian histone demethylase, e.g. LSD1 orLSD2.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in certain aspects, their recovery,purification, and use for one or more of the purposes disclosed herein.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

For example, a “C1-C3 alkyl” group can be selected from methyl, ethyl,n-propyl, i-propyl, and cyclopropyl, or from a subset thereof. Incertain aspects, the “C1-C3 alkyl” group can be optionally furthersubstituted. As a further example, a “C1-C4 alkyl” group can be selectedfrom methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl,s-butyl, t-butyl, and cyclobutyl, or from a subset thereof. In certainaspects, the “C1-C4 alkyl” group can be optionally further substituted.As a further example, a “C1-C6 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl,cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane,neohexane, and cyclohexane, or from a subset thereof. In certainaspects, the “C1-C6 alkyl” group can be optionally further substituted.As a further example, a “C1-C8 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl,cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane,neohexane, cyclohexane, heptane, cycloheptane, octane, and cyclooctane,or from a subset thereof. In certain aspects, the “C1-C8 alkyl” groupcan be optionally further substituted. As a further example, a “C1-C12alkyl” group can be selected from methyl, ethyl, n-propyl, i-propyl,cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl,i-pentyl, s-pentyl, t-pentyl, neopentyl, cyclopentyl, n-hexyl, i-hexyl,3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane,cycloheptane, octane, cyclooctane, nonane, cyclononane, decane,cyclodecane, undecane, cycloundecane, dodecane, and cyclododecane, orfrom a subset thereof. In certain aspects, the “C1-C12 alkyl” group canbe optionally further substituted.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” or “haloalkyl” specifically refers to analkyl group that is substituted with one or more halide, e.g., fluorine,chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refersto an alkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol as described herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(a)—, where “a” is an integer of from2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as -OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as -OA¹-OA² or-OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, nitrite,sulfonamide, or thiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, nitrile, sulfonamide, or thiol asdescribed herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol, as described herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, nitrile, sulfonamide, or thiol asdescribed herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula -NA¹A², where A¹ and A² can be, independently, hydrogen oralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “alkylamino” as used herein is represented by the formula—NH(-alkyl) where alkyl is a described herein. Representative examplesinclude, but are not limited to, methylamino group, ethylamino group,propylamino group, isopropylamino group, butylamino group, isobutylaminogroup, (sec-butyl)amino group, (tert-butyl)amino group, pentylaminogroup, isopentylamino group, (tert-pentyl)amino group, hexylamino group,and the like.

The term “dialkylamino” as used herein is represented by the formula—N(-alkyl)₂ where alkyl is a described herein. Representative examplesinclude, but are not limited to, dimethylamino group, diethylaminogroup, dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)aminogroup, dipentylamino group, diisopentylamino group, di(tert-pentyl)aminogroup, dihexylamino group, N-ethyl-N-methylamino group,N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “polyester” as used herein is represented by the formula-(A¹O(O)C-A²-C(O)O)_(a)— or -(A¹O(O)C-A²-OC(O))_(a)—, where A¹ and A²can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and“a” is an integer from 1 to 500. “Polyester” is as the term used todescribe a group that is produced by the reaction between a compoundhaving at least two carboxylic acid groups with a compound having atleast two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group describedherein. The term “polyether” as used herein is represented by theformula -(A¹O-A²O)_(a)-, where A¹ and A² can be, independently, analkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group described herein and “a” is an integer of from 1 to500. Examples of polyether groups include polyethylene oxide,polypropylene oxide, and polybutylene oxide.

The terms “halogen,” “halide,” and “halo,” as used herein, refer to thehalogens fluorine, chlorine, bromine, and iodine. It is alsocontemplated that, in various aspects, halogen can be selected fromfluoro, chloro, bromo, and iodo. For example, halogen can be selectedfrom fluoro, chloro, and bromo. As a further example, halogen can beselected from fluoro and chloro. As a further example, halogen can beselected from chloro and bromo. As a further example, halogen can beselected from bromo and iodo. As a further example, halogen can beselected from chloro, bromo, and iodo. In one aspect, halogen can befluoro. In a further aspect, halogen can be chloro. In a still furtheraspect, halogen is bromo. In a yet further aspect, halogen is iodo.

It is also contemplated that, in certain aspects, pseudohalogens (e.g.triflate, mesylate, tosylate, brosylate, etc.) can be used in place ofhalogens. For example, in certain aspects, halogen can be replaced bypseudohalogen. As a further example, pseudohalogen can be selected fromtriflate, mesylate, tosylate, and brosylate. In one aspect,pseudohalogen is triflate. In a further aspect, pseudohalogen ismesylate. In a further aspect, pseudohalogen is tosylate. In a furtheraspect, pseudohalogen is brosylate.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes azetidine, dioxane,furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole,including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran,tetrazine, including 1,2,4,5-tetrazine, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole,thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine,triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group asdescribed herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or an alkyl,cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen oran alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, or heteroaryl group as described herein. Throughout thisspecification “S(O)” is a short hand notation for S═O. The term“sulfonyl” is used herein to refer to the sulfo-oxo group represented bythe formula —S(O)₂A′, where A¹ can be hydrogen or an alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupas described herein. The term “sulfone” as used herein is represented bythe formula A'S(O)₂A², where A¹ and A² can be, independently, an alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein. The term “sulfoxide” as usedherein is represented by the formula A¹S(O)A², where A¹ and A² can be,independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. In is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘); —(CH₂)₀₋₄(O)SR^(∘);—(CH₂)₀₋₄(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘); —OC(O)(CH₂)₀₋₄SR—,SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂;—C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)_(o)OC(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘);—C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘);—(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘);—P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straightor branched)alkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•); -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•)), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•)), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄straight or branched alkylene)C(O)OR^(•), or —SSR^(•) wherein each R^(•)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₄₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(•), -(haloR^(•)), —OH, —OR^(•); —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(•); -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —ON,—C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein eachR^(•) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

The term “leaving group” refers to an atom (or a group of atoms) withelectron withdrawing ability that can be displaced as a stable species,taking with it the bonding electrons. Examples of suitable leavinggroups include halides—including chloro, bromo, and iodo—andpseudohalides (sulfonate esters)—including triflate, mesylate, tosylate,and brosylate. It is also contemplated that a hydroxyl moiety can beconverted into a leaving group via Mitsunobu reaction.

The terms “hydrolysable group” and “hydrolysable moiety” refer to afunctional group capable of undergoing hydrolysis, e.g., under basic oracidic conditions. Examples of hydrolysable residues include, withoutlimitation, acid halides, activated carboxylic acids, and variousprotecting groups known in the art (see, for example, Protective Groupsin Organic Synthesis, T. W. Greene, P. G. M. Wuts, Wiley-Interscience,1999).

The term “protecting group” means a group which protects one or morefunctional groups of a compound giving rise to a protected derivative ofthe specified compound. Functional groups which may be protectedinclude, by way of example, amino groups, hydroxyl groups, and the like.Protecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group, include, but are notlimited to, tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl(Cbz), 9-fluorenylmethoxycarbonyl (FMOC), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), benzyl, p-methoxybenzyl, p-fluorobenzyl,p-chlorobenzyl, p-bromobenzyl, diphenylmethyl naphtylmethyl,tetrahydropyran (THP), and the like.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(1-6C)-alkylsilyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS), and the like; esters(acyl groups) including (1-6C)-alkanoyl groups, such as formyl, acetyl,and the like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM),tetrahydropyran (THP), methoxylmethyl (MOM), methylthiomethyl (MTM),benzyloxymethyl (BOM), and the like.

The term “organic residue” defines a carbon containing residue, i.e., aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. In a further aspect, an organic residuecan comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbonatoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

A very close synonym of the term “residue” is the term “radical,” whichas used in the specification and concluding claims, refers to afragment, group, or substructure of a molecule described herein,regardless of how the molecule is prepared. For example, a2,4-thiazolidinedione radical in a particular compound has thestructure:

regardless of whether thiazolidinedione is used to prepare the compound.In some embodiments the radical (for example an alkyl) can be furthermodified (i.e., substituted alkyl) by having bonded thereto one or more“substituent radicals.” The number of atoms in a given radical is notcritical to the present invention unless it is indicated to the contraryelsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain oneor more carbon atoms. An organic radical can have, for example, 1-26carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms,1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organicradical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbonatoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organicradicals often have hydrogen bound to at least some of the carbon atomsof the organic radical. One example, of an organic radical thatcomprises no inorganic atoms is a 5,6,7,8-tetrahydro-2-naphthyl radical.In some embodiments, an organic radical can contain 1-10 inorganicheteroatoms bound thereto or therein, including halogens, oxygen,sulfur, nitrogen, phosphorus, and the like. Examples of organic radicalsinclude but are not limited to an alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, mono-substituted amino, di-substituted amino,acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substitutedalkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide,alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy,substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl,heteroaryl, heterocyclic, or substituted heterocyclic radicals, whereinthe terms are defined elsewhere herein. A few non-limiting examples oforganic radicals that include heteroatoms include alkoxy radicals,trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals andthe like.

“Inorganic radicals,” as the term is defined and used herein, contain nocarbon atoms and therefore comprise only atoms other than carbon.Inorganic radicals comprise bonded combinations of atoms selected fromhydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, andhalogens such as fluorine, chlorine, bromine, and iodine, which can bepresent individually or bonded together in their chemically stablecombinations. Inorganic radicals have 10 or fewer, or preferably one tosix or one to four inorganic atoms as listed above bonded together.Examples of inorganic radicals include, but not limited to, amino,hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonlyknown inorganic radicals. The inorganic radicals do not have bondedtherein the metallic elements of the periodic table (such as the alkalimetals, alkaline earth metals, transition metals, lanthanide metals, oractinide metals), although such metal ions can sometimes serve as apharmaceutically acceptable cation for anionic inorganic radicals suchas a sulfate, phosphate, or like anionic inorganic radical. Inorganicradicals do not comprise metalloids elements such as boron, aluminum,gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gaselements, unless otherwise specifically indicated elsewhere herein.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or l meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture.

Many of the compounds described herein can have one or more chiralcenters and therefore can exist in different enantiomeric forms. Ifdesired, a chiral carbon can be designated with an asterisk (*). Whenbonds to the chiral carbon are depicted as straight lines in thedisclosed formulas, it is understood that both the (R) and (S)configurations of the chiral carbon, and hence both enantiomers andmixtures thereof, are embraced within the formula. As is used in theart, when it is desired to specify the absolute configuration about achiral carbon, one of the bonds to the chiral carbon can be depicted asa wedge (bonds to atoms above the plane) and the other can be depictedas a series or wedge of short parallel lines is (bonds to atoms belowthe plane). The Cahn-Inglod-Prelog system can be used to assign the (R)or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labelled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl,respectively. Compounds further comprise prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labelled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labelled compounds of the present invention and prodrugsthereof can generally be prepared by carrying out the procedures below,by substituting a readily available isotopically labelled reagent for anon-isotopically labelled reagent.

The compounds described in the invention can be present as a solvate. Insome cases, the solvent used to prepare the solvate is an aqueoussolution, and the solvate is then often referred to as a hydrate. Thecompounds can be present as a hydrate, which can be obtained, forexample, by crystallization from a solvent or from aqueous solution. Inthis connection, one, two, three or any arbitrary number of solvate orwater molecules can combine with the compounds according to theinvention to form solvates and hydrates. Unless stated to the contrary,the invention includes all such possible solvates.

The term “co-crystal” means a physical association of two or moremolecules which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g. “Crystal Engineering of the Composition ofPharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a NewPath to Improved Medicines?” Almarasson, O., et. al., The Royal Societyof Chemistry, 1889-1896, 2004. Examples of co-crystals includep-toluenesulfonic acid and benzenesulfonic acid.

It is also appreciated that certain compounds described herein can bepresent as an equilibrium of tautomers. For example, ketones with anα-hydrogen can exist in an equilibrium of the keto form and the enolform.

Likewise, amides with an N-hydrogen can exist in an equilibrium of theamide form and the imidic acid form. Unless stated to the contrary, theinvention includes all such possible tautomers.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

In some aspects, a structure of a compound can be represented by aformula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R^(n) is understood torepresent five independent substituents, R^(n(a)), R^(n(b)), R^(n(c)),R^(n(d)), R^(n(e)). By “independent substituents,” it is meant that eachR substituent can be independently defined. For example, if in oneinstance R^(n(a)) is halogen, then R^(n(b)) is not necessarily halogenin that instance.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Sigma-Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. COMPOUNDS

In one aspect, the invention relates to compounds useful as inhibitorsof histone demethylase. In a further aspect, the compounds are useful asinhibitors of lysine-specific histone demethylase (“LSD”). Moreover, inone aspect, the compounds of the invention are useful in the treatmentof disorders of uncontrolled cellular proliferations. In a furtheraspect, the disorder of uncontrolled cellular proliferation is a canceror a tumor. In a still further aspect, the disorder of uncontrolledcellular proliferation is associated with a LSD dysfunction, as furtherdescribed herein.

It is contemplated that each disclosed derivative can be optionallyfurther substituted. It is also contemplated that any one or morederivative can be optionally omitted from the invention. It isunderstood that a disclosed compound can be provided by the disclosedmethods. It is also understood that the disclosed compounds can beemployed in the disclosed methods of using.

1. Structure

In one aspect, the invention relates to a compound having a structurerepresented by formula (I)

or a formula (II)

-   -   wherein    -   m is 0 or 1;    -   n is an integer from 0 to 3;    -   X is selected from the group consisting of OH, NO₂ and F;    -   Z is selected from the group consisting of N and CH;    -   R₁ is selected from the group consisting of halo, C1-C3        haloalkyl, and C1-C3 polyhaloalkyl;    -   each of R₂, R₃, and R₄ is independently selected from the group        consisting of hydrogen, halo, hydroxyl, cyano, amino, C2-C6        alkalkoxy, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 polyhaloalkyl, and        C1-C6 haloalkyl;    -   R₅ is selected from the group consisting of NR₆ R₇, C1-C6 alkyl,        C3-C6 cycloalkyl,

-   -   and Cy, and substituted with 0-3 groups independently selected        from halo, hydroxyl, amino, C2-C6 alkalkoxy, C1-C6 alkylalcohol,        C1-C6 alkoxy, C1-C6 alkyl, C1-C6 polyhaloalkyl, C1-C6 haloalkyl,        C3-C6 cycloalkyl, and Cy;    -   Cy is a heterocycloalkyl selected from the group consisting of        aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl,        oxazolidinyl, imidazolidinyl, pyrazolidinyl, piperazinyl,        oxazinanyl, morpholinyl, hexahydrophyrimidinyl, and        hexahydropyridazinyl; and    -   each of R₆ and R₇ is independently selected from the group        consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, and C3-C6        heterocycloalkyl;    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a compound selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.

In yet other embodiments, the invention provides a compound having astructure represented by a formula:

or a pharmaceutically acceptable salt thereof.

In yet other embodiments, the invention provides a compound having astructure represented by a formula:

or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of any of a compound of the inventionand a pharmaceutically acceptable carrier.

The invention also provides a method for the treatment of a disorder ofuncontrolled cellular proliferation in a mammal, the method comprisingthe step of administering to the mammal an effective amount of any ofthe compounds of the invention.

The invention also provides a method for decreasing histone demethylaseactivity in a mammal, the method comprising the step of administering tothe mammal an effective amount of any of the compounds of the invention.

2. Inhibition of Histone Demethylase Activity

In one aspect, the disclosed compounds exhibit inhibition of LSD proteinactivity. In a yet further aspect, the disclosed compounds exhibitselective inhibition of LSD1 protein activity. In an even furtheraspect, the disclosed compounds exhibit selective inhibition of LSD2protein activity. In a still further aspect, the disclosed compoundsinhibit LSD demethylase activity. In a still further aspect, thedisclosed compounds exhibit binding to the FAD domain of LSD. In an evenfurther aspect, the disclosed compounds exhibit inhibition ofLSD-mediated demethylation of histone 3 (H3) at the Lys4 position. In astill further aspect, the disclosed compounds exhibit inhibitionLSD-mediated demethylation of H3K3m1 and H3K4me2. In a yet furtheraspect, the disclosed compounds exhibit inhibition LSD-mediateddemethylation of H3K9me2 and H3K9me1.

In a still further aspect, the disclosed compounds inhibit LSD1demethylase activity. In a still further aspect, the disclosed compoundsexhibit binding to the FAD domain of LSD1. In an even further aspect,the disclosed compounds exhibit inhibition of LSD1-mediateddemethylation of histone 3 (H3) at the Lys4 position. In a still furtheraspect, the disclosed compounds exhibit inhibition LSD1-mediateddemethylation of H3K3m1 and H3K4me2. In a yet further aspect, thedisclosed compounds exhibit inhibition LSD1-mediated demethylation ofH3K9me2 and H3K9me1.

In a still further aspect, the disclosed compounds inhibit LSD2demethylase activity. In a still further aspect, the disclosed compoundsexhibit binding to the FAD domain of LSD2. In an even further aspect,the disclosed compounds exhibit inhibition of LSD2-mediateddemethylation of histone 3 (H3) at the Lys4 position. In a still furtheraspect, the disclosed compounds exhibit inhibition LSD2-mediateddemethylation of H3K3m1 and H3K4me2.

In a further aspect, the disclosed compounds exhibit disruption of LSDinteraction with a complexes comprising one or more of HDAC1/2, CoREST,CtBP1, BRAF35 and BHC80 proteins. In a still further aspect, thedisclosed compounds disrupt binding of LSD1 to one or more proteinsselected from HDAC1/2, CoREST, CtBP1, BRAF35 and BHC80 proteins. In astill further aspect, the disclosed compounds disrupt binding of LSD2 toone or more proteins selected from G9a, NSD3, HDAC1/2, CoREST, CtBP1,BRAF35 and BHC80 proteins.

Inhibition of LSD activity can be determined by a variety of both invitro and in vivo methods known to one skilled in the art. For example,enzymatic activity can be determined in in vitro enzyme assay systems.In various aspects, the enzymatic activity of either LSD1 or LSD2 can bedetermined in a spectrophometric assay. Briefly, the assay is based onthe multistep enzymatic reaction in which LSD1 or LSD2 first producesH₂O₂ during the demethylation of lysine 4 on a peptide corresponding tothe first 21 amino acids of the N-terminal tail of histone H3. In thepresence of horseradish peroxidase, the H₂O₂ produced reacts with ADHPto produce the highly fluorescent compound resorufin that can beanalyzed with an excitation wavelength of 530-540 nm and an emissionwavelength of 585-595 nm. The assay requires a source of LSD1 or LSD2enzyme, either purified from natural sources (e.g. a tissue or culturedcells), isolated as a recombinantly expressed protein, or as aunpurified protein in whole cell extracts. In one aspect, the disclosedcompounds exhibit inhibition of LSD protein activity with an IC₅₀ in anEMSA assay of less than about 300 μM, less than about 100 μM, less thanabout 50 μM, less than about 10 μM, less than about 1 μM, less thanabout 500 nM, or of less than about 100 nM. In a further aspect, thedisclosed compounds exhibit inhibition of LSD1 protein activity with anIC₅₀ in an EMSA assay of less than about 300 μM, less than about 100less than about 50 μM, less than about 10 μM, less than about 1 μM, lessthan about 500 nM, or of less than about 100 nM. In a still furtheraspect, the disclosed compounds exhibit inhibition of LSD2 proteinactivity with an IC₅₀ in an EMSA assay of less than about 300 μM, lessthan about 100 μM, less than about 50 μM, less than about 10 μM, lessthan about 1 μM, less than about 500 nM, or of less than about 100 nM.

In one aspect, the disclosed compounds are selective for LSD. In afurther aspect, selective inhibition of LSD activity is determined usingan enzyme assay. In various further aspects, the compound inhibits LSDactivity in an enzyme assay with an IC₅₀ less than the IC₅₀ for MAO Aand/or MAO B. That is, a disclosed compound can have selectivity for theLSD protein vis-à-vis MAO A and/or MAO B. For example, in one aspect, adisclosed compound can inhibit LSD with an IC₅₀ of about 5-fold lessthan that for MAO A, of about 10-fold less than that for MAO A, of about20-fold less than that for MAO A, of about 30-fold less than that forMAO A, of about 50-fold less than that for MAO A, of about 100-fold lessthan that for MAO A, of about 250-fold less than that for MAO A, ofabout 500-fold less than that for MAO A, of about 1000-fold less thanthat for MAO A, and more than about 1000-fold less than that for MAO A.In a further aspect, a disclosed compound can inhibit LSD with an IC₅₀of about 5-fold less than that for MAO B, of about 10-fold less thanthat for MAO B, of about 20-fold less than that for MAO B, of about30-fold less than that for MAO B, of about 50-fold less than that forMAO B, of about 100-fold less than that for MAO B, of about 250-foldless than that for MAO B, of about 500-fold less than that for MAO B, ofabout 1000-fold less than that for MAO B, and more than about 1000-foldless than that for MAO B.

In one aspect, the disclosed compounds are selective for LSD1. In afurther aspect, selective inhibition of LSD1 activity is determinedusing an enzyme assay. In various further aspects, the compound inhibitsLSD1 activity in an enzyme assay with an IC₅₀ less than the IC₅₀ for oneor more of LSD2, MAO A, and MAO B. That is, a disclosed compound canhave selectivity for the LSD1 protein vis-à-vis one or more of LSD2, MAOA, and MAO B. For example, in one aspect, a disclosed compound caninhibit LSD1 with an IC₅₀ of about 5-fold less than that for LSD2, ofabout 10-fold less than that for LSD2, of about 20-fold less than thatfor LSD2, of about 30-fold less than that for LSD2, or of about 50-foldless than that for LSD2. In a further aspect, a disclosed compound caninhibit LSD1 with an IC₅₀ of about 5-fold less than that for MAO A, ofabout 10-fold less than that for MAO A, of about 20-fold less than thatfor MAO A, of about 30-fold less than that for MAO A, of about 50-foldless than that for MAO A, of about 100-fold less than that for MAO A, ofabout 250-fold less than that for MAO A, of about 500-fold less thanthat for MAO A, of about 1000-fold less than that for MAO A, and morethan about 1000-fold less than that for MAO A. In a further aspect, adisclosed compound can inhibit LSD1 with an IC₅₀ of about 5-fold lessthan that for MAO B, of about 10-fold less than that for MAO B, of about20-fold less than that for MAO B, of about 30-fold less than that forMAO B, of about 50-fold less than that for MAO B, of about 100-fold lessthan that for MAO B, of about 250-fold less than that for MAO B, ofabout 500-fold less than that for MAO B, of about 1000-fold less thanthat for MAO B, and more than about 1000-fold less than that for MAO B.

In one aspect, the disclosed compounds are selective for LSD2. In afurther aspect, selective inhibition of LSD2 activity is determinedusing an enzyme assay. In various further aspects, the compound inhibitsLSD2 activity in an enzyme assay with an IC₅₀ less than the IC₅₀ for oneor more of LSD1, MAO A, and MAO B. That is, a disclosed compound canhave selectivity for the LSD2 protein vis-à-vis one or more of LSD1, MAOA, and MAO B. For example, in one aspect, a disclosed compound caninhibit LSD2 with an IC₅₀ of about 5-fold less than that for LSD1, ofabout 10-fold less than that for LSD1, of about 20-fold less than thatfor LSD1, of about 30-fold less than that for LSD1, or of about 50-foldless than that for LSD1. In a further aspect, a disclosed compound caninhibit LSD2 with an IC₅₀ of about 5-fold less than that for MAO A, ofabout 10-fold less than that for MAO A, of about 20-fold less than thatfor MAO A, of about 30-fold less than that for MAO A, of about 50-foldless than that for MAO A, of about 100-fold less than that for MAO A, ofabout 250-fold less than that for MAO A, of about 500-fold less thanthat for MAO A, of about 1000-fold less than that for MAO A, and morethan about 1000-fold less than that for MAO A. In a further aspect, adisclosed compound can inhibit LSD2 with an IC₅₀ of about 5-fold lessthan that for MAO B, of about 10-fold less than that for MAO B, of about20-fold less than that for MAO B, of about 30-fold less than that forMAO B, of about 50-fold less than that for MAO B, of about 100-fold lessthan that for MAO B, of about 250-fold less than that for MAO B, ofabout 500-fold less than that for MAO B, of about 1000-fold less thanthat for MAO B, and more than about 1000-fold less than that for MAO B.

In various aspects, the disclosed compounds exhibit binding to a LSDprotein. In a further aspect, the disclosed compounds exhibit binding tothe FAD domain of a LSD protein. In a still further aspect, thedisclosed compounds exhibit binding to LSD1 protein. In an even furtheraspect, the disclosed compounds exhibit binding to LSD2 protein. Thebinding affinity of a disclosed compound for a LSD protein, e.g. LSD1protein, can be determined by various methods known to one skilled inthe art. In one aspect, the disclosed compounds exhibit binding to LSDprotein with a K_(D) of less than about 50 μM, less than about 10 μM,less than about 1 μM, less than about 500 nM, or of less than about 100nM. In a further aspect, the K_(D) is determined using an SPR method. Ina still further aspect, the binding is determined using LSD1 protein. Ina yet further aspect, the binding is determined using LSD2 protein.

In various further aspects, the binding to LSD is selective. In afurther aspect, the disclosed compounds exhibit a K_(D) for LSD bindingless than the K_(D) of MAO A and/or MAO B. That is, a disclosed compoundcan have selectivity for the LSD protein vis-à-vis MAO A and/or MAO Bproteins. For example, in one aspect, a disclosed compound can bind LSDwith a K_(D) of about 5-fold less than that for MAO A, of about 10-foldless than that for MAO A, of about 20-fold less than that for MAO A, ofabout 30-fold less than that for MAO A, of about 50-fold less than thatfor MAO A, of about 100-fold less than that for MAO A, of about 250-foldless than that for MAO A, of about 500-fold less than that for MAO A, ofabout 1000-fold less than that for MAO A, and of more than about1000-fold less than that for MAO A. In a further aspect, a disclosedcompound can bind LSD with a K_(D) of about 5-fold less than that forMAO B, of about 10-fold less than that for MAO B, of about 20-fold lessthan that for MAO B, of about 30-fold less than that for MAO B, of about50-fold less than that for MAO B, of about 100-fold less than that forMAO B, of about 250-fold less than that for MAO B, of about 500-foldless than that for MAO B, of about 1000-fold less than that for MAO B,and of more than about 1000-fold less than that for MAO B.

In various further aspects, the binding to LSD1 is selective. In afurther aspect, the disclosed compounds exhibit a K_(D) for LSD1 bindingless than the K_(D) for one or more of LSD2, MAO A, and MAO B. That is,a disclosed compound can have selectivity for the LSD1 protein vis-à-visone or more of LSD2, MAO A, and MAO B proteins. For example, in oneaspect, a disclosed compound can bind LSD1 with a K_(D) of about 5-foldless than that for LSD2, of about 10-fold less than that for LSD2, ofabout 20-fold less than that for LSD2, of about 30-fold less than thatfor LSD2, or of about 50-fold less than that for LSD2. In a furtheraspect, a disclosed compound can bind LSD1 with a K_(D) of about 5-foldless than that for MAO A, of about 10-fold less than that for MAO A, ofabout 20-fold less than that for MAO A, of about 30-fold less than thatfor MAO A, of about 50-fold less than that for MAO A, of about 100-foldless than that for MAO A, of about 250-fold less than that for MAO A, ofabout 500-fold less than that for MAO A, of about 1000-fold less thanthat for MAO A, and of more than about 1000-fold less than that for MAOA. In a further aspect, a disclosed compound can bind LSD1 with a K_(D)of about 5-fold less than that for MAO B, of about 10-fold less thanthat for MAO B, of about 20-fold less than that for MAO B, of about30-fold less than that for MAO B, of about 50-fold less than that forMAO B, of about 100-fold less than that for MAO B, of about 250-foldless than that for MAO B, of about 500-fold less than that for MAO B, ofabout 1000-fold less than that for MAO B, and of more than about1000-fold less than that for MAO B.

In various further aspects, the binding to LSD2 is selective. In afurther aspect, the disclosed compounds exhibit a K_(D) for LSD2 bindingless than the K_(D) for one or more of LSD1, MAO A, and MAO B. That is,a disclosed compound can have selectivity for the LSD2 protein vis-à-visone or more of LSD1, MAO A, and MAO B proteins. For example, in oneaspect, a disclosed compound can bind LSD2 with a K_(D) of about 5-foldless than that for LSD1, of about 10-fold less than that for LSD1, ofabout 20-fold less than that for LSD1, of about 30-fold less than thatfor LSD1, or of about 50-fold less than that for LSD1. In a furtheraspect, a disclosed compound can bind LSD2 with a K_(D) of about 5-foldless than that for MAO A, of about 10-fold less than that for MAO A, ofabout 20-fold less than that for MAO A, of about 30-fold less than thatfor MAO A, of about 50-fold less than that for MAO A, of about 100-foldless than that for MAO A, of about 250-fold less than that for MAO A, ofabout 500-fold less than that for MAO A, of about 1000-fold less thanthat for MAO A, and of more than about 1000-fold less than that for MAOA. In a further aspect, a disclosed compound can bind LSD2 with a K_(D)of about 5-fold less than that for MAO B, of about 10-fold less thanthat for MAO B, of about 20-fold less than that for MAO B, of about30-fold less than that for MAO B, of about 50-fold less than that forMAO B, of about 100-fold less than that for MAO B, of about 250-foldless than that for MAO B, of about 500-fold less than that for MAO B, ofabout 1000-fold less than that for MAO B, and of more than about1000-fold less than that for MAO B.

Alternatively, the inhibition of STAT protein activity can be determinedin a cell-based assay. There are a variety of cell-based assays that aresuitable for determination of inhibition of LSD protein activity knownto one skilled in the art. For example, cell growth inhibition or cellarrest can be determined using a cell, either a permanent cell-line or aprimary cell culture that has a LSD protein with dysfunction activity.In a further aspect, the LSD protein is LSD1. In a still further aspect,the LSD protein is LSD2. In a yet further aspect, the LSD proteindysfunction is one wherein the LSD protein is has acquired a gain offunction mutation. Alternatively, the LSD protein dysfunction has aphenotype of persistent or constitutive activity. For example, the LSDprotein can have a persistent or constitutive activity due to adysfunction in an upstream regulatory protein. In a further aspect, theLSD protein is overexpressed due to a dysfunction in regulation oftranscription and/or translation of the LSD gene. In a further aspect,the cell harbors an active oncogene is associated with LSD dysfunction.

In one aspect, the disclosed compounds and products of disclosed methodsof making inhibit cell growth. In a still further aspect, the disclosedcompounds and products of disclosed methods inhibit cell growth in an invitro assay system. In an even further aspect, the in vitro assay systemmakes use of a cell-line derived from a from cancer or tumor selectedfrom breast cancer, ovarian cancer, testicular cancer, lung cancer,liver cancer, prostate cancer, pancreatic cancer and a sarcoma. In a yetfurther aspect, the cell-line is derived from a human source. In a yetfurther aspect, the disclosed compounds inhibit cell growth in a cellwith a persistently active LSD protein. In an even further aspect, thecell-line has an activated LSD protein. In a still further aspect, thecell-line is selected from AN3 CA, BT-20, BT-549, HCT 116, HER218, MCF7,MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468, PANC-1, PC-3, SK-N-MC,T-47D, and U-87 MG. In one aspect, the disclosed compounds exhibitinhibition of cell growth activity in an in vitro cell-based assay withan IC₅₀ of less than about 500 μM, of less than about about 250 μM, lessthan about 100 μM, less than about 50 μM, less than about 10 μM, lessthan about 1 μM, less than about 500 nM, of less than about 100 nM, ofless than about 10 nM, and of less than about 1 nM.

In one aspect, the disclosed compounds and products of disclosed methodsof making inhibit cell migration. In a still further aspect, thedisclosed compounds and products of disclosed methods inhibit cellmigration in an in vitro assay system. In an even further aspect, the invitro assay system makes use of a cell-line derived from a from canceror tumor selected from breast cancer, ovarian cancer, testicular cancer,lung cancer, liver cancer, prostate cancer, pancreatic cancer and asarcoma. In a yet further aspect, the cell-line is derived from a humansource. In a yet further aspect, the disclosed compounds inhibit cellgrowth in a cell with a persistently active LSD protein. In an evenfurther aspect, the cell-line has an activated LSD protein. In a stillfurther aspect, the cell-line is selected from AN3 CA, BT-20, BT-549,HCT 116, HER218, MCF7, MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468,PANC-1, PC-3, SK-N-MC, T-47D, and U-87 MG. In one aspect, the disclosedcompounds exhibit inhibition of cell migration in an in vitro cell-basedassay with an IC₅₀ of less than about 300 less than about 100 μM, lessthan about 50 μM, less than about 10 OA less than about 1 μM, less thanabout 500 nM, or of less than about 100 nM.

C. METHODS OF MAKING THE COMPOUNDS

In one aspect, the invention relates to methods of making compoundsuseful as inhibitors of LSD. In a further aspect, the products ofdisclosed methods of making are modulators of LSD activity. In a yetfurther aspect, the products of disclosed methods of making bind to aSTAT protein and negatively modulate LSD activity. The compounds can, inone aspect, exhibit subtype selectivity. In a still further aspect, theproducts of disclosed methods of making exhibit selectivity for the LSD1member of the LSD protein family. In an even further aspect, theproducts of the disclosed methods of making exhibit selectivity for theLSD2 member of the LSD protein family.

In one aspect, the invention relates to methods of making compoundsuseful as inhibitors of histone demethylase, which can be useful in thetreatment of disorders of uncontrolled cellular proliferation. In afurther aspect, the histone demethylase is LSD1. In a yet furtheraspect, the histone demethylase is LSD2.

The compounds of this invention can be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature, exemplified in theexperimental sections or clear to one skilled in the art. For clarity,examples having a single substituent are shown where multiplesubstituents are allowed under the definitions disclosed herein.

Reactions used to generate the compounds of this invention are preparedby employing reactions as shown in the following Reaction Schemes, inaddition to other standard manipulations known in the literature or toone skilled in the art. The following examples are provided so that theinvention might be more fully understood, are illustrative only, andshould not be construed as limiting.

In one aspect, the disclosed compounds comprise the products of thesynthetic methods described herein. In a further aspect, the disclosedcompounds comprise a compound produced by a synthetic method describedherein. In a still further aspect, the invention comprises apharmaceutical composition comprising a therapeutically effective amountof the product of the disclosed methods and a pharmaceuticallyacceptable carrier. In a still further aspect, the invention comprises amethod for manufacturing a medicament comprising combining at least onecompound of any of disclosed compounds or at least one product of thedisclosed methods with a pharmaceutically acceptable carrier or diluent.

1. Route I

In one aspect, substituted (E)-N′-(1-phenylethylidene)benzohydrazideanalogs of the present invention can be prepared generically by thesynthetic scheme as shown below.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein. A more specific example is setforth below.

In one aspect, Route I begins with a suitable substituted acidderivative (1.1). Suitable substituted acid derivatives (1.1) arecommercially available or can be readily prepared by one skilled in theart. In a typical reaction, compound of the type 1.1 is added to theamine derivative of type 1.2 in the presence of a suitable base, e.g.,potassium carbonate, in suitable solvent such as THF. The reaction isstirred at room temperature (about 15-30° C.) for a time sufficient tocomplete the reaction, e.g., about twelve hours. After completion of thereaction, the solvent is removed under vacuum, and the compound of type1.3 is isolated and purified by chromatography.

In one aspect, compounds of type 1.4 can be prepared by reaction ofcompounds of type 1.3 with an alcohol by an esterification reaction. Ina typical reaction, a compound of type 1.3 is heated at a suitabletemperature (e.g., at reflux, about 65° C.) in a suitable alcoholicsolvent, e.g., methanol, in the presence of an acid catalyst such asconcentrated sulfuric acid for a time sufficient to complete thereaction, e.g., overnight (about 8-18 h). After completion of thereaction, the solvent is removed under vacuum, and the compound of type1.4, is isolated and purified by chromatography.

In one aspect, compounds of type 1.4 can provide compounds of type 1.5by reaction with an appropriate hydrazine derivative (NH2NHR4). In atypical reaction, a compound of type 1.4 is added to a suitablehydrazine derivative (NH2NHR4) and heated at suitable temperature (e.g.,at reflux, about 65° C.) in a suitable solvent, e.g., methanol, for atime sufficient to complete the reaction (e.g., about 12 h). Aftercompletion of the reaction, the solvent is removed under vacuum, and thecompound of type 1.5, is isolated and purified by chromatography.

In one aspect, compounds of type 1.5 can provide compounds of type 1.7by reaction with an appropriate carbonyl-containing compound (1.6). In atypical reaction, a compound of type 1.6 and a suitable hydrazinederivative (1.5) are dissolved in a suitable solvent, e.g., methanol, inthe presence of a suitable acid catalyst (e.g., acetic acid), and themixture is heated using a microwave reactor at suitable temperature,e.g., about 120° C., for a time sufficient to complete the reaction(e.g., about 30 min). After completion of the reaction and followingcooling, the solvent is removed under vacuum, and the compounds of type1.7, are isolated and purified by chromatography.

2. Route II

In one aspect, substituted (E)-N′-(1-phenylethylidene)benzohydrazideanalogs of the present invention can be prepared generically by thesynthetic scheme as shown below.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein. A more specific example is setforth below.

In one aspect, Route II begins with a suitable substituted acidderivative (2.1). Suitable substituted acid derivatives (2.1) arecommercially available or can be readily prepared by one skilled in theart. In one aspect, compounds of type 2.2 can be prepared by reaction ofcompounds of type 2.1 with an alcohol by an esterification reaction. Ina typical reaction, a compound of type 2.1 is heated at a suitabletemperature (e.g., at reflux, about 70° C.) in a suitable alcoholicsolvent, e.g., methanol, in the presence of an acid catalyst such asconcentrated sulfuric acid for a time sufficient to complete thereaction, e.g., overnight (about 8-18 h). After completion of thereaction, the solvent is removed under vacuum, and the compound of type2.2, is isolated and purified by chromatography.

In one aspect, compounds of type 2.2 can provide compounds of type 2.3by reaction with an appropriate hydrazine derivative (NH₂NHR⁴). In atypical reaction, a compound of type 2.2 is added to a suitablehydrazine derivative (NH₂NHR⁴) and heated a suitable temperature (e.g.,at reflux, about 70° C.) in a suitable solvent, e.g., methanol for timesufficient to complete the reaction such as overnight (8-18 h). Aftercompletion of the reaction, the solvent is removed under vacuum, and thecompound of type 2.3, is isolated and purified by chromatography.

In one aspect, compounds of type 2.3 can be used to provide compounds oftype 2.5 by reaction with an appropriate carbonyl-containing compound(2.4). In a typical reaction, a compound of type 2.4 and a suitablehydrazine derivative (2.3) are dissolved in a suitable solvent, e.g.,methanol, in the presence of a suitable acid catalyst (e.g., aceticacid), and the mixture is heated using a microwave reactor at suitabletemperature, e.g., about 120° C., at a time sufficient to complete thereaction (e.g., about 30 min). After completion of the reaction andfollowing cooling, the solvent is removed under vacuum, and thecompounds of type 2.5, are isolated and purified by chromatography.

In a further aspect, the compound produced exhibits inhibition of ahistone demethylase. In a still further aspect, the histone demethylaseis a member of the lysine-specific (“LSD”) family of histonedemethylases. In yet further aspect, the histone demethylase is LSD1. Inan even further aspect, the histone demethylase is LSD2. In a stillfurther aspect, the compound produced exhibits inhibition of cellviability.

In a further aspect, the compound produced exhibits inhibition with anIC₅₀ of less than about 1.0×10⁻⁴ M. In a still further aspect, thecompound produced exhibits inhibition with an IC₅₀ of less than about1.0×10⁻⁵ M. In a yet further aspect, the compound produced exhibitsinhibition with an IC₅₀ of less than about 1.0×10⁻⁶ M. In an evenfurther aspect, the compound produced exhibits inhibition with an IC₅₀of less than about 1.0×10⁻⁷ M. In a still further aspect, the compoundproduced exhibits inhibition with an IC₅₀ of less than about 1.0×10⁻⁸ M.In a yet further aspect, the compound produced exhibits inhibition withan IC₅₀ of less than about 1.0×10⁻⁹ M.

It is contemplated that each disclosed methods can further compriseadditional steps, manipulations, and/or components. It is alsocontemplated that any one or more step, manipulation, and/or componentcan be optionally omitted from the invention.

It is understood that a disclosed methods can be used to provide thedisclosed compounds. It is also understood that the products of thedisclosed methods can be employed in the disclosed methods of using.

D. PHARMACEUTICAL COMPOSITIONS

In one aspect, the invention relates to pharmaceutical compositionscomprising the disclosed compounds. That is, a pharmaceuticalcomposition can be provided comprising a therapeutically effectiveamount of at least one disclosed compound or at least one product of adisclosed method and a pharmaceutically acceptable carrier.

In a further aspect, the invention relates to pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and aneffective amount of the product of a disclosed synthetic method. In afurther aspect, the effective amount is a therapeutically effectiveamount. In a further aspect, the effective amount is a prophylacticallyeffective amount. In a further aspect, the compound is a disclosedcompound.

In certain aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids”,includes inorganic acids, organic acids, and salts prepared therefrom,for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier can take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof, can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds of the invention. The compounds of theinvention, or pharmaceutically acceptable salts thereof, can also beincluded in pharmaceutical compositions in combination with one or moreother therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, dusting powder, mouth washes, gargles, and the like.Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereof,via conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in moulds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

In the treatment conditions which require inhibition or negativemodulation of LSD protein activity an appropriate dosage level willgenerally be about 0.01 to 500 mg per kg patient body weight per day andcan be administered in single or multiple doses. Preferably, the dosagelevel will be about 0.1 to about 250 mg/kg per day; more preferably 0.5to 100 mg/kg per day. A suitable dosage level can be about 0.01 to 250mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kgper day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or5.0 to 50 mg/kg per day. For oral administration, the compositions arepreferably provided in the from of tablets containing 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and1000 milligrams of the active ingredient for the symptomatic adjustmentof the dosage of the patient to be treated. The compound can beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day. This dosing regimen can be adjusted to provide theoptimal therapeutic response.

It is understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors. Such factorsinclude the age, body weight, general health, sex, and diet of thepatient. Other factors include the time and route of administration,rate of excretion, drug combination, and the type and severity of theparticular disease undergoing therapy.

The present invention is further directed to a method for themanufacture of a medicament for inhibiting or negatively modulating LSDprotein activity (e.g., treatment of a disorder of uncontrolled cellularproliferation, or one or more neurodegenerative disorders associatedwith LSD dysfunction) in mammals (e.g., humans) comprising combining oneor more disclosed compounds, products, or compositions with apharmaceutically acceptable carrier or diluent. Thus, in one aspect, theinvention relates to a method for manufacturing a medicament comprisingcombining at least one disclosed compound or at least one disclosedproduct with a pharmaceutically acceptable carrier or diluent.

The disclosed pharmaceutical compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above mentioned pathological conditions.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

E. METHODS OF USING THE COMPOUNDS AND COMPOSITIONS

The disclosed compounds can be used as single agents or in combinationwith one or more other drugs in the treatment, prevention, control,amelioration or reduction of risk of the aforementioned diseases,disorders and conditions for which compounds of formula I or the otherdrugs have utility, where the combination of drugs together are safer ormore effective than either drug alone. The other drug(s) can beadministered by a route and in an amount commonly used therefore,contemporaneously or sequentially with a disclosed compound. When adisclosed compound is used contemporaneously with one or more otherdrugs, a pharmaceutical composition in unit dosage form containing suchdrugs and the disclosed compound is preferred. However, the combinationtherapy can also be administered on overlapping schedules. It is alsoenvisioned that the combination of one or more active ingredients and adisclosed compound will be more efficacious than either as a singleagent.

The pharmaceutical compositions and methods of the present invention canfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of the above mentionedpathological conditions.

1. Treatment Methods

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety of disorderswherein the patient or subject would benefit from inhibition or negativemodulation of a LSD protein. In one aspect, a treatment can includeselective inhibition of LSD to an extent effective to affect histonedemethylation activity. Thus, a disorder can be associated with histonedemethylation activity, for example dysfunctional epigenetic regulationof genes in a cancer cell. In one aspect, provided is a method oftreating or preventing a disorder in a subject comprising the step ofadministering to the subject at least one disclosed compound; at leastone disclosed pharmaceutical composition; and/or at least one disclosedproduct in a dosage and amount effective to treat the disorder in thesubject.

Also provided is a method for the treatment of one or more disorders,for which LSD inhibition is predicted to be beneficial, in a subjectcomprising the step of administering to the subject at least onedisclosed compound; at least one disclosed pharmaceutical composition;and/or at least one disclosed product in a dosage and amount effectiveto treat the disorder in the subject.

In one aspect, provided is a method for treating a disorder ofuncontrolled cellular proliferation, comprising: administering to asubject at least one disclosed compound; at least one disclosedpharmaceutical composition; and/or at least one disclosed product in adosage and amount effective to treat the disorder in the subject. In afurther aspect, provided is a method for treating or preventing aneurodegenerative disorder, comprising: administering to a subject atleast one disclosed compound; at least one disclosed pharmaceuticalcomposition; and/or at least one disclosed product in a dosage andamount effective to treat the disorder in the subject. Also provided isa method for the treatment of a disorder in a mammal comprising the stepof administering to the mammal at least one disclosed compound,composition, or medicament.

The invention is directed at the use of described chemical compositionsto treat diseases or disorders in patients (preferably human) whereinLSD inhibition would be predicted to have a therapeutic effect, such asdisorders of uncontrolled cellular proliferation (e.g. cancers) andneurodegenerative disorders such as Alzheimer's disease, Huntington'sdisease, and Parkinson's disease, by administering one or more disclosedcompounds or products.

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety of disordersof uncontrolled cellular proliferation. In one aspect, the disorder ofuncontrolled cellular proliferation is associated with a histonedemethylase dysfunction. In a further aspect, the histone demethylasedysfunction is disregulation of the LSD. In a still further aspect, thehistone demethylase dysfunction is disregulation of the LSD1. In an evenfurther aspect, the histone demethylase dysfunction is disregulation ofthe LSD2.

Also provided is a method of use of a disclosed compound, composition,or medicament. In one aspect, the method of use is directed to thetreatment of a disorder. In a further aspect, the disclosed compoundscan be used as single agents or in combination with one or more otherdrugs in the treatment, prevention, control, amelioration or reductionof risk of the aforementioned diseases, disorders and conditions forwhich the compound or the other drugs have utility, where thecombination of drugs together are safer or more effective than eitherdrug alone. The other drug(s) can be administered by a route and in anamount commonly used therefore, contemporaneously or sequentially with adisclosed compound. When a disclosed compound is used contemporaneouslywith one or more other drugs, a pharmaceutical composition in unitdosage form containing such drugs and the disclosed compound ispreferred. However, the combination therapy can also be administered onoverlapping schedules. It is also envisioned that the combination of oneor more active ingredients and a disclosed compound can be moreefficacious than either as a single agent.

Examples of disorders associated with a histone demethylase dysfunctioninclude a disorder of uncontrolled cellular proliferation. In a yetfurther aspect, the disorder of uncontrolled cellular proliferation iscancer. In a yet further aspect, the cancer is a leukemia. In an evenfurther aspect, the cancer is a sarcoma. In a still further aspect, thecancer is a solid tumor. In a yet further aspect, the cancer is alymphoma.

It is understood that cancer refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. The cancer may be multi-drug resistant (MDR) or drug-sensitive.Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include breast cancer, prostate cancer, colon cancer, squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, cervical cancer, ovariancancer, peritoneal cancer, liver cancer, e.g., hepatic carcinoma,bladder cancer, colorectal cancer, endometrial carcinoma, kidney cancer,and thyroid cancer.

In various aspects, further examples of cancers are basal cellcarcinoma, biliary tract cancer; bone cancer; brain and CNS cancer;choriocarcinoma; connective tissue cancer; esophageal cancer; eyecancer; cancer of the head and neck; gastric cancer; intra-epithelialneoplasm; larynx cancer; lymphoma including Hodgkin's and Non-Hodgkin'slymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g.,lip, tongue, mouth, and pharynx); retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas

In a further aspect, the cancer is a hematological cancer. In a stillfurther aspect, the hematological cancer is selected from acute myeloidleukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloidleukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia(JMML), Hodgkin lymphoma, Non-Hodgkin lymphoma, multiple myeloma,solitary myeloma, localized myeloma, and extramedullary myeloma. In astill further aspect, the cancer is selected from chronic lymphocyticleukemia, small lymphocytic lymphoma, B-cell non-Hodgkin lymphoma, andlarge B-cell lymphoma.

In a further aspect, the cancer is a cancer of the brain. In a stillfurther aspect, the cancer of the brain is selected from a glioma,medulloblastoma, primitive neuroectodermal tumor (PNET), acousticneuroma, glioma, meningioma, pituitary adenoma, schwannoma, CNSlymphoma, primitive neuroectodermal tumor, craniopharyngioma, chordoma,medulloblastoma, cerebral neuroblastoma, central neurocytoma,pineocytoma, pineoblastoma, atypical teratoid rhabdoid tumor,chondrosarcoma, chondroma, choroid plexus carcinoma, choroid plexuspapilloma, craniopharyngioma, dysembryoplastic neuroepithelial tumor,gangliocytoma, germinoma, hemangioblastoma, hemangiopercytoma, andmetastatic brain tumor. In a yet further aspect, the glioma is selectedfrom ependymoma, astrocytoma, oligodendroglioma, and oligoastrocytoma.In an even further aspect, the glioma is selected from juvenilepilocytic astrocytoma, subependymal giant cell astrocytoma,ganglioglioma, subependymoma, pleomorphic xanthoastrocytom, anaplasticastrocytoma, glioblastoma multiforme, brain stem glioma,oligodendroglioma, ependymoma, oligoastrocytoma, cerebellar astrocytoma,desmoplastic infantile astrocytoma, subependymal giant cell astrocytoma,diffuse astrocytoma, mixed glioma, optic glioma, gliomatosis cerebri,multifocal gliomatous tumor, multicentric glioblastoma multiforme tumor,paraganglioma, and ganglioglioma.

In one aspect, the cancer can be a cancer selected from cancers of theblood, brain, genitourinary tract, gastrointestinal tract, colon,rectum, breast, kidney, lymphatic system, stomach, lung, pancreas, andskin. In a further aspect, the cancer is selected from prostate cancer,glioblastoma multiforme, endometrial cancer, breast cancer, and coloncancer. In a further aspect, the cancer is selected from a cancer of thebreast, ovary, prostate, head, neck, and kidney. In a still furtheraspect, the cancer is selected from cancers of the blood, brain,genitourinary tract, gastrointestinal tract, colon, rectum, breast,livery, kidney, lymphatic system, stomach, lung, pancreas, and skin. Ina yet further aspect, the cancer is selected from a cancer of the lungand liver. In an even further aspect, the cancer is selected from acancer of the breast, ovary, testes and prostate In a still furtheraspect, the cancer is a cancer of the breast. In a yet further aspect,the cancer is a cancer of the ovary. In an even further aspect, thecancer is a cancer of the prostate. In a still further aspect, thecancer is a cancer of the testes.

In various aspects, disorders associated with a histone demethylasedysfunction include neurodegenerative disorders. In a further aspect,the neurodegenerative disease is selected from Alzheimer's disease,Parkinson's disease, and Huntington's disease.

The compounds are further useful in a method for the prevention,treatment, control, amelioration, or reducation of risk of the diseases,disorders and conditions noted herein. The compounds are further usefulin a method for the prevention, treatment, control, amelioration, orreduction of risk of the aforementioned diseases, disorders andconditions in combination with other agents.

The present invention is further directed to administration of a LSDinhibitor for improving treatment outcomes in the context of disordersof uncontrolled cellular proliferation, including cancer. That is, inone aspect, the invention relates to a cotherapeutic method comprisingthe step of administering to a mammal an effective amount and dosage ofat least one compound of the invention in connection with cancertherapy.

In a further aspect, administration improves treatment outcomes in thecontext of cancer therapy. Administration in connection with cancertherapy can be continuous or intermittent. Administration need not besimultaneous with therapy and can be before, during, and/or aftertherapy. For example, cancer therapy can be provided within 1, 2, 3, 4,5, 6, 7 days before or after administration of the compound. As afurther example, cancer therapy can be provided within 1, 2, 3, or 4weeks before or after administration of the compound. As a still furtherexample, cognitive or behavioral therapy can be provided before or afteradministration within a period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 half-lives of the administered compound.

In one aspect, the disclosed compounds can be used in combination withone or more other drugs in the treatment, prevention, control,amelioration, or reduction of risk of diseases or conditions for whichdisclosed compounds or the other drugs can have utility, where thecombination of the drugs together are safer or more effective thaneither drug alone. Such other drug(s) can be administered, by a routeand in an amount commonly used therefor, contemporaneously orsequentially with a compound of the present invention. When a compoundof the present invention is used contemporaneously with one or moreother drugs, a pharmaceutical composition in unit dosage form containingsuch other drugs and a disclosed compound is preferred. However, thecombination therapy can also include therapies in which a disclosedcompound and one or more other drugs are administered on differentoverlapping schedules. It is also contemplated that when used incombination with one or more other active ingredients, the disclosedcompounds and the other active ingredients can be used in lower dosesthan when each is used singly.

Accordingly, the pharmaceutical compositions include those that containone or more other active ingredients, in addition to a compound of thepresent invention.

The above combinations include combinations of a disclosed compound notonly with one other active compound, but also with two or more otheractive compounds. Likewise, disclosed compounds can be used incombination with other drugs that are used in the prevention, treatment,control, amelioration, or reduction of risk of the diseases orconditions for which disclosed compounds are useful. Such other drugscan be administered, by a route and in an amount commonly used therefor,contemporaneously or sequentially with a compound of the presentinvention. When a compound of the present invention is usedcontemporaneously with one or more other drugs, a pharmaceuticalcomposition containing such other drugs in addition to a disclosedcompound is preferred. Accordingly, the pharmaceutical compositionsinclude those that also contain one or more other active ingredients, inaddition to a compound of the present invention.

The weight ratio of a disclosed compound to the second active ingredientcan be varied and will depend upon the effective dose of eachingredient. Generally, an effective dose of each will be used. Thus, forexample, when a compound of the present invention is combined withanother agent, the weight ratio of a disclosed compound to the otheragent will generally range from about 1000:1 to about 1;1000, preferablyabout 200:1 to about 1:200. Combinations of a compound of the presentinvention and other active ingredients will generally also be within theaforementioned range, but in each case, an effective dose of each activeingredient should be used.

In such combinations a disclosed compound and other active agents can beadministered separately or in conjunction. In addition, theadministration of one element can be prior to, concurrent to, orsubsequent to the administration of other agent(s).

Accordingly, the subject compounds can be used alone or in combinationwith other agents which are known to be beneficial in the subjectindications or other drugs that affect receptors or enzymes that eitherincrease the efficacy, safety, convenience, or reduce unwanted sideeffects or toxicity of the disclosed compounds. The subject compound andthe other agent can be coadministered, either in concomitant therapy orin a fixed combination.

In one aspect, the compound can be employed in combination withanti-cancer therapeutic agents or other known therapeutic agents.

In the treatment of conditions which require inhibition or negativemodulation of LSD, an appropriate dosage level will generally be about0.01 to 1000 mg per kg patient body weight per day which can beadministered in single or multiple doses. Preferably, the dosage levelwill be about 0.1 to about 250 mg/kg per day; more preferably about 0.5to about 100 mg/kg per day. A suitable dosage level can be about 0.01 to250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 01 to 50mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5or 5 to 50 mg/kg per day. For oral administration, the compositions arepreferably provided in the form of tablets containing 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and1000 milligrams of the active ingredient for the symptomatic adjustmentof the dosage to the patient to be treated. The compounds can beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day. This dosage regimen can be adjusted to provide theoptimal therapeutic response. It will be understood, however, that thespecific dose level and frequency of dosage for any particular patientcan be varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy.

Thus, in one aspect, the invention relates to methods for inhibiting ornegatively modulating LSD in at least one cell, comprising the step ofcontacting the at least one cell with at least one compound of theinvention, in an amount effective to modulate or activate LSD activityresponse, e.g. LSD1 or LSD2, in the at least one cell. In a furtheraspect, the cell is mammalian, for example human. In a further aspect,the cell has been isolated from a subject prior to the contacting step.In a further aspect, contacting is via administration to a subject.

a. Treatment of a Disorder of Uncontrolled Cellular Proliferation

In one aspect, the invention relates to a method for the treatment of adisorder of uncontrolled cellular proliferation in a mammal, the methodcomprising the step of administering to the mammal an effective amountof least one disclosed compound or a product of a disclosed method ofmaking a compound, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof, thereby treating the disorder ofuncontrolled cellular proliferation.

In a still further aspect, the effective amount is a therapeuticallyeffective amount. In a yet still further aspect, the effective amount isa prophylactically effective amount.

In a further aspect, the mammal is a human. In a yet further aspect, themethod further comprises the step of identifying a mammal in need oftreatment of a disorder of uncontrolled cellular proliferation. In astill further aspect, the mammal has been diagnosed with a need fortreatment of a disorder of uncontrolled cellular proliferation prior tothe administering step.

In a further aspect, the disorder of uncontrolled cellular proliferationis associated with a histone demethylase dysfunction. In a furtheraspect, the histone demethylase is a lysine-specific histonedemethylase. In a yet further aspect, the lysine-specific histonedemethylase is LSD1. In an even further aspect, the lysine-specifichistone demethylase is LSD2.

In a further aspect, the disorder of uncontrolled cellular proliferationis a cancer. In a yet further aspect, the cancer is a leukemia. In aneven further aspect, the cancer is a sarcoma. In a still further aspect,the cancer is a solid tumor. In a yet further aspect, the cancer is alymphoma. In an even further aspect, the cancer is selected from chroniclymphocytic leukemia, small lymphocytic lymphoma, B-cell non-Hodgkinlymphoma, and large B-cell lymphoma. In a still further aspect, thecancer is selected from cancers of the blood, brain, genitourinarytract, gastrointestinal tract, colon, rectum, breast, livery, kidney,lymphatic system, stomach, lung, pancreas, and skin. In a yet furtheraspect, the cancer is selected from a cancer of the lung and liver. Inan even further aspect, the cancer is selected from a cancer of thebreast, ovary, testes and prostate. In a still further aspect, thecancer is a cancer of the breast. In a yet further aspect, the cancer isa cancer of the ovary. In an even further aspect, the cancer is a cancerof the prostate. In a still further aspect, the cancer is a cancer ofthe testes.

b. Decreasing Histone Demethylase Activity

In one aspect, the invention relates to a method for decreasing histonedemethylase activity in a mammal, the method comprising the step ofadministering to the mammal an effective amount of at least onedisclosed compound or a product of a disclosed method of making acompound, or a pharmaceutically acceptable salt, hydrate, solvate, orpolymorph thereof, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof, thereby decreasing histone demethylaseactivity in the mammal.

In a still further aspect, the effective amount is a therapeuticallyeffective amount. In a yet still further aspect, the effective amount isa prophylactically effective amount.

In a further aspect, the mammal is a human. In a yet further aspect, themethod further comprises the step of identifying a mammal in need ofdecreasing histone demethylase activity. In a still further aspect, themammal has been diagnosed with a need for decreasing histone demethylaseactivity prior to the administering step.

In a further aspect, the histone demethylase is a lysine-specifichistone demethylase. In a yet further aspect, the lysine-specifichistone demethylase is LSD1. In an even further aspect, thelysine-specific histone demethylase is LSD2.

In a further aspect, the need for decreasing histone demethylaseactivity is associated with a histone demethylase dysfunction. In a yetfurther aspect, the histone demethylase dysfunction is associated with adisorder of uncontrolled cellular proliferation. In a yet furtheraspect, the method further comprises the step of identifying a mammal inneed of treating a disorder of uncontrolled cellular proliferation. In astill further aspect, the mammal has been diagnosed with a need fortreating a disorder of uncontrolled cellular proliferation prior to theadministering step.

In a still further aspect, the disorder of uncontrolled cellularproliferation is a cancer. In a yet further aspect, the cancer is aleukemia. In an even further aspect, the cancer is a sarcoma. In a stillfurther aspect, the cancer is a solid tumor. In a yet further aspect,the cancer is a lymphoma. In an even further aspect, the cancer isselected from chronic lymphocytic leukemia, small lymphocytic lymphoma,B-cell non-Hodgkin lymphoma, and large B-cell lymphoma. In a stillfurther aspect, the cancer is selected from cancers of the blood, brain,genitourinary tract, gastrointestinal tract, colon, rectum, breast,livery, kidney, lymphatic system, stomach, lung, pancreas, and skin. Ina yet further aspect, the cancer is selected from a cancer of the lungand liver. In an even further aspect, the cancer is selected from acancer of the breast, ovary, testes and prostate. In a still furtheraspect, the cancer is a cancer of the breast. In a yet further aspect,the cancer is a cancer of the ovary. In an even further aspect, thecancer is a cancer of the prostate. In a still further aspect, thecancer is a cancer of the testes.

c. Decreasing Histone Demethylase Activity in Cells

In one aspect, the invention relates to a method for decreasing histonedemethylase activity in at least one cell, the method comprising thestep of contacting the at least one cell with an effective amount ofleast one disclosed compound or a product of a disclosed method ofmaking a compound, or a pharmaceutically acceptable salt, hydrate,solvate, or polymorph thereof, or a pharmaceutically acceptable salt,hydrate, solvate, or polymorph thereof, thereby decreasing histonedemethylase activity in the cell.

In a still further aspect, the effective amount is a therapeuticallyeffective amount. In a yet still further aspect, the effective amount isa prophylactically effective amount.

In a further aspect, the cell is mammalian. In a still further aspect,the cell is human. In a yet further aspect, contacting is viaadministration to a mammal. In a further aspect, the method furthercomprises the step of identifying the mammal as having a need ofdecreasing histone demethylase activity in a cell. In a still furtheraspect, the mammal has been diagnosed with a need for decreasing histonedemethylase activity prior to the administering step.

In a further aspect, the histone demethylase is a lysine-specifichistone demethylase. In a yet further aspect, the lysine-specifichistone demethylase is LSD1. In an even further aspect, thelysine-specific histone demethylase is LSD2.

In a further aspect, the need for decreasing histone demethylaseactivity in a cell is associated with a disorder of uncontrolledcellular. In a still further aspect, the disorder of uncontrolledcellular proliferation is a cancer. In a yet further aspect, the canceris a leukemia. In an even further aspect, the cancer is a sarcoma. In astill further aspect, the cancer is a solid tumor. In a yet furtheraspect, the cancer is a lymphoma. In an even further aspect, the canceris selected from chronic lymphocytic leukemia, small lymphocyticlymphoma, B-cell non-Hodgkin lymphoma, and large B-cell lymphoma. In astill further aspect, the cancer is selected from cancers of the blood,brain, genitourinary tract, gastrointestinal tract, colon, rectum,breast, livery, kidney, lymphatic system, stomach, lung, pancreas, andskin. In a yet further aspect, the cancer is selected from a cancer ofthe lung and liver. In an even further aspect, the cancer is selectedfrom a cancer of the breast, ovary, testes and prostate. In a stillfurther aspect, the cancer is a cancer of the breast. In a yet furtheraspect, the cancer is a cancer of the ovary. In an even further aspect,the cancer is a cancer of the prostate. In a still further aspect, thecancer is a cancer of the testes.

2. Manufacture of a Medicament

In one aspect, the invention relates to a method for the manufacture ofa medicament for inhibition of histone demethylase activity in a mammalcomprising combining a therapeutically effective amount of a disclosedcompound or product of a disclosed method with a pharmaceuticallyacceptable carrier or diluent.

F. EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Several methods for preparing the compounds of this invention areillustrated in the following Examples. Starting materials and therequisite intermediates are in some cases commercially available, or canbe prepared according to literature procedures or as illustrated herein.

The following exemplary compounds of the invention were synthesized. TheExamples are provided herein to illustrate the invention, and should notbe construed as limiting the invention in any way. The Examples aretypically depicted in free base form, according to the IUPAC namingconvention. However, some of the Examples were obtained or isolated insalt form.

As indicated, some of the Examples were obtained as racemic mixtures ofone or more enantiomers or diastereomers. The compounds may be separatedby one skilled in the art to isolate individual enantiomers. Separationcan be carried out by the coupling of a racemic mixture of compounds toan enantiomerically pure compound to form a diastereomeric mixture,followed by separation of the individual diastereomers by standardmethods, such as fractional crystallization or chromatography. A racemicor diastereomeric mixture of the compounds can also be separateddirectly by chromatographic methods using chiral stationary phases.

1. General Chemical Materials and Methods

All analytical or anhydrous grade reagents were purchased fromcommercial sources and were used without further purification. Solventswere of analytical or anhydrous grade (Sigma-Aldrich). Specialtychemicals and building blocks obtained from several suppliers were ofthe highest offered purity (always 95%).

NMR spectroscopy was performed on a Varian Unity 400 instrument with a 5mm broadband probe and using standard pulse sequences. Chemical shifts(δ) are reported in parts-per-million (ppm) downfield from solventreferences. Coupling constants (J-values) are expressed in Hz.

Mass spectrometry was performed on a Finnigan LCQ Duo LCMS ion trapelectrospray (ESI) mass spectrometer. All samples were analyzed bypositive ESI-MS and the mass-to-charge ratio (m/z) of the protonatedmolecular ion is reported.

Microwave-assisted reactions were performed on a Biotage Initiator 2.5at various powers.

Hydrogenation reactions were performed on a standard Parr hydrogenationapparatus.

Reactions were monitored either by HPLC or TLC. When monitored by TLC,reactions were analyzed on Baker flexible-backed plates coated with 200μm of silica gel containing a fluorescent indicator. Preparative TLC wasperformed on 20 cm×20 cm. Analtech Uniplates coated with a 1000 or 2000μm silica gel layer containing a fluorescent (UV 254) indicator. Elutionmixtures are reported as v:v. Spot visualization was achieved using UVlight.

Flash chromatography was performed on a Teledyne Isco CombiFlash RF 200using appropriately sized Redisep Rf Gold or Standard normal-phasesilica or reversed-phase C-18 columns. Crude compounds were adsorbed onsilica gel, 70-230 mesh 40 Å (for normal phase) or Celite 503 (forreversed-phase) and loaded into solid cartridges. Elution mixtures arereported as v:v.

2. Molecular Modeling and Virtual Screening Methods

All computational studies employed PDB ID 2Z5U for the structuralcoordinates of LSD1. Virtual docking methods ICM, Glide and GOLDprograms were implemented. The protein structure was prepared by 3Dprotonation, deletion of water molecules and energy minimization usingthe ICM force field and distance-dependent dielectric potential with anRMS gradient of 0.1; heavy atoms in the protein were kept fixed, andhistidine residues were considered as neutral. Virtual screeningcalculations utilized default parameters (unless explicitly specifiedotherwise) with ICM and Glide scores as scoring functions respectively.In both cases, FAD was defined as the ligand and an active site regionwas defined by a sphere of radius 12 Å around the bound FAD in complexwith LSD1.

Confirmation of the accuracy and efficiency of the applied dockingprotocol used the FAD cofactor adenine dinucleotide fragment, and theflavin fragment, and known LSD1 inhibitors (decoy set) as positivecontrols. Two separate docking runs were carried out with ICM and theGlide docking program; GOLD docking was employed for re-scoring.

The compound database was prepared using Ligprep 2.1.23 (Schrödinger,LLC., New York, N.Y.). Two rounds of VS, including HTVS and standardprecision (SP) docking, were adopted. The top 10000 compounds ranked byGlide were stored and submitted for additional docking experiments usingICM docking. The final set of 2000 hits was selected based on ICM scoresand individual compounds were visually inspected to check the dockingposes and interactions between ligands and LSD1. GOLD consensus scoringfunctions were employed further to re-score these 2000 hits selectedfrom Glide and ICM. Finally, 121 compounds were purchased (if available)or synthesized for LSD1 inhibition studies.

3. MD Simulation Methods

All simulations were performed using the AMBER ff99SB force field(Hornak, V., et al. Proteins 2006, 65 (3), 712-25) for LSD1, the generalAmber force field (′gaff′; see Wang, J., et al. J Comput Chem 2004, 25(9), 1157-74) for compound 12, and the TIP3P (Jorgensen, W. L., Journalof Chemical Physics 1982, (77), 4156-4163) model for water was employed.The simulations approximated long-range electrostatic interactions usingthe particle-mesh Ewald method (PME) procedure (Essmann, U., et al.Journal of Chemical Physics 1995, (103), 8577-8593; Darden, T., et al.Journal of Chemical Physics 1993, (98), 10089-10092). Using LEaP thebinding modes generated from ICM docking in complex with LSD1 weresolvated to neutral charge and the complexes were first minimized withPMEMD (Case, D. A, et al. AMBER11, San Francisco, 2010). Followingminimization, 200 ps of unrestrained molecular dynamics simulation usinga non-bonded interaction cutoff of 9 Å was run for both binding modeswith a constant pressure periodic boundary maintaining 1 atm of pressureand isotropic position scaling with a relaxation time of 2 ps. SHAKE wasused to constrain bonds involving hydrogen and Langevin dynamics wereused to regulate temperature (Case, D. A., et al. AMBER11, SanFrancisco, 2010), maintaining 300 K. Relative free energies of bindingfor comparisons between the two binding modes were predicted usingMMPBSA.py⁹ with 100 snapshots at 1-ps intervals starting either at 1 psor 101 ps into the trajectory.

4. Virtual Screening Results

The first crystal structures of LSD1 elucidating critical architecturalfeatures were later by Stavropoulos et al. (Nat Struct Mol Biol 2006,13(7):626-32; Protein Data Bank or PDB ID 2H94; seehttp://www.wwpdb.org/), Yang et al. (Mol Cell 2006, 23 (3), 377-87; PDBID 2IW5), and Chen et al. (Proc Natl Acad Sci USA 2006, 103 (38),13956-61; PDB ID 2HKO). These 2.9 Å, 2.57 Å, and 2.8 Å structures,respectively, show a highly negatively charged substrate-binding cavityspacious enough to accommodate the N-terminal tail of histone H3.Further, an N-terminal SWIRM domain and an insertion in the corecatalytic domain, termed the Tower Domain, were established as necessarystructural motifs for enzymatic activity and interactions with cofactorssuch as CoREST. For the studies described herein, the structure, PDB ID2Z5U, was used with bound LSD1 inhibitor tranylcypromine forcomputational studies, including virtual screening, docking, andmolecular dynamics (Mimasu, S., et al. Biochem Biophys Res Commun 2008,366 (1), 15-22). In order to evaluate the chemical space outside oftranylcypromine and polyamine derivatives, HTVS was used with anin-house library. The library was curated from publicly available vendorlibraries, totaling approximately 13 million compounds, using customfilters developed in-house. Compounds were filtered based on Lipinski'srule of five, with exceptions occurring in only 62,000 compounds.Further, structurally redundant compounds were removed such that theresulting library contained a diverse, yet manageable set of about 2million compounds. Prior to screening, compounds were prepared using theLigPrep module of the Schrodinger Suite as well as ICM's inbuiltpreparation of three-dimensional (3D) ligands such that physiologicallyrelevant protonation states were used.

Prepared ligands were then docked against three different sites on LSD1;the FAD site located in the amine oxidase domain, and the adeninedinucleotide and flavin fragments of this pocket. The docking protocolsused by both ICM and Glide were run with the FAD, adenine dinucleotide,flavin fragments, and known LSD1 inhibitors to check for accuracy. Inaddition to the docking algorithm rankings, visual inspection of thedocking results was used to evaluate binding position, suitable pose,and orientation. Taken together, the scoring functions from ICM andGlide were able to correctly identify known inhibitors within the top 2%of the decoy set used. GOLD was used to re-score and the GOLD fitnessfunction produced similar enrichments.

A virtual screen was set up against the FAD-binding pocket of LSD1 usingthe established docking protocol and the 2 million-compound database.The top 10,000 compounds were selected from both ICM and Glide scoringfunctions for further analysis. A few identical compounds were scoredsimilarly between the two algorithms; this redundancy was filtered out.Furthermore, visual inspection was performed to filter out similarcompounds and to increase the diversity of the final selection. Visualanalysis also allowed identification of key interactions within theFAD-binding pocked of LSD1. These include hydrogen bonding with Ser289,Arg310 and Arg316, van der Waals interactions with Va1590 and Leu625,and π interactions with Trp756. Moreover, compounds with hydroxyl andhydrophobic electron withdrawing groups seemed to show increasedenrichment in the initial docking results. The FAD-binding pocket ofLSD1 is a deep and narrow crevice in the interior of the protein and issurrounded by hydrophobic amino acid residues. Thus the hydrophobiccharacter of the compounds may play an important role in the random walkof the compound into the active site.

Based on the selection criteria discussed above, 121 structurallydistinct compounds were procured and submitted for biochemical screeningagainst LSD1. The biochemical assay, as described in the experimentalsection, measures H₂O₂ produced from the oxidative demethylation of apeptide substrate. From the 121 compounds, a series of relatedcompounds, which showed potent activity in the biochemical assay, wereidentified. Docking scores, ranks, and accompanying biochemical assayresults for the series are presented in Tables 1, 2, and 6-9.

Of the ten active compounds in Table 1 (and associated tables providingbiochemical and cellular data, Tables 6, 8, and 9), that were discoveredusing virtual screening methods, e.g. compounds 1, 2, 4 and 5 showedsimilar binding modes within the FAD-binding site of LSD1. Additionally,the docking scores for compounds 1, 2, 4 and 5 correlated well with theobserved biochemical activity. These results suggested that improvedinhibitors targeted toward the adenine dinucleotide pocket in the amineoxidase domain of LSD1 were accessible.

The Glide scores are predictive and correlated well with compoundshaving p-OH or m-Cl aryl substitutions (compounds 1 and 5). It is clearfrom these studies that the hydrophobic electron withdrawing groups suchas —Cl are tolerated, whereas small alkyl substituents such as methyl(e.g. compound 8) or fused bicyclic containing compound 10 have loweractivity. Introduction of any donating groups particularly the —OCH₃functional group at the 2^(nd) position lost activity due to lack ofGly314 H-bonding interactions (e.g. compound 6). The lack of biochemicalactivity of compound 6 was highly predictive from docking scores, whereICM and Glide provided −18.39 and −6.63 kcal/mol energies respectively.In subsequent docking analysis, additional benzohydrazine series ofcompounds were identified, with hydrazine —C methyl or aryl4-substituted sulfone containing compounds, as exemplified by thevirtual hit compound 9, which exhibited potent LSD1 inhibition activitywith an IC₅₀ of 19 nM. The low docking score of compound 9 is primarilydue to the shift in 2-OH aryl ring position. Compound 9, with asulfone/morpholine substituent, was chosen as a backbone for furtheroptimization due, in part, to its chemical stability.

The binding mode of compound 12 with the sulfone/morpholine is depictedwith the docking pose predicted from ICM in FIG. 1. In this model, thephenolic group fits well in the pocket composed of residues Ser289,Gly314 and Arg316. The central carbonyl group appears to be involved instrong H-bonding interactions with Arg310 amino group and the morpholineoxygen shows H-bonding interactions with Va1590. These sets of hydrogenbonding interactions were also observed with Glide and GOLD dockingexperiments. The additional experiments showed the morpholinesubstituted aryl ring participating in Tr-Tr interactions with Trp756residue while the morpholine oxygen retained in H-bonding with Va1590.

Chemical optimization also focused on the design of compounds containingheteroaryl rings on either side of compound 12. Computational modelsusing these results generated a variety of chemically plausiblescaffolds, from which a substituted pyridine was identified as being anappropriate moiety capable of interacting with Ser289, Gly314 andArg316, surrounding residues and ideal properties. A representative iscompound 24, which had potent LSD1 activity of (28 nM) and alsoexhibited a similar binding mode to that of compound 12 (see FIG. 2).

Many of the representative compounds contain a C-alkyl hydrazine toincrease metabolic stability of the series. However, a bulkier group,like the ethyl group of compound 21, isn't well accommodated by thebinding pocket as illustrated in different biochemical activities ofcompounds 12 and 21. Aryl substitution with methylsulfone (compound 25)and substituted with a morpholine ring (compound 12) increasedbiochemical efficacy by roughly an order of magnitude when compared tocompound 11. Addition of only a morpholine ring maintains somebiochemical activity as illustrated by compound 23. Replacing thesulfono-morpholine with sulfono-N-dimethyl also maintained biochemicalactivity as illustrated by compound 18. Additionally, replacement of the2-OH group with a chloro was found not to be well accommodated and asignificant drop in activity was demonstrated between compounds 12 and16. Results with compound 24 suggest that using a substituted pyridineis accommodated by the enzyme, but various other substitutions andheterocycles generally resulted in a drop in biochemical activity asillustrated in compounds 13, 14, 15, 17, 19, 20 and 22.

Many of the representative compounds in Table 2 contained a C-alkylhydrazine to increase metabolic stability of the series. However, abulkier group, e.g. the ethyl group of compound 21, is less wellaccommodated by the binding pocket as illustrated in the differentbiochemical activities of compounds 12 and 21. The aryl substitutionwith methylsulfone (e.g. compound 25) and substituted with a morpholinering (compound 12) increased biochemical efficacy by roughly an order ofmagnitude when compared to compound 11. Addition of a heterocycle, e.g.a morpholine ring, maintains biochemical activity as illustrated bycompound 23. Replacing the sulfono-morpholine with sulfono-N-dimethylalso maintained biochemical activity as illustrated by compound 18.Additionally, replacement of the 2-OH group with a chloro was found notto be accommodated with a significant drop in activity between compounds12 and 16. As discussed above, compound 24 suggests that using asubstituted pyridine is accommodated by the enzyme. Further analysissuggests that the hydroxyl of compound 12 is associated with increasedbiochemical activity, e.g. when this substituent group is substitutedwith a chlorine (compound 16), activity was decreased.

TABLE 1 ICM Glide Gold No. Structure Score Score Fitness  1

−42.25 −8.14 56.26  2

−42.25 −7.92 58.21  3

−21.91 −7.87 51.29  4

−37.77 −8.64 57.69  5

−36.3 −8.84 47.98  6

−18.39 −6.63 49.93  7

−8.16 −7.21 41.86  8

−8.5 −6.81 52.19  9

−24 −6.26 43.26 10

−20.97 −6.14 46.64

TABLE 2 ICM Glide Gold No. Structure Score Score Fitness 11

−29.76 −7.89 58.21 12

−38.16 −8.96 58.17 13

−36.14 −9.21 54.88 14

−23.81 −6.75 46.21 15

−31.24 −7.91 51.29 16

−41.26 −6.87 53.29 17

−29.23 −7.93 43.29 18

−41.96 −9.87 53.92 19

−27.24 −6.87 43.76 20

−21.41 −6.28 37.28 21

−23.11 −7.21 39.84 22

−19.88 −6.97 37.24 23

−38.11 −8.21 46.81 24

−37.11 −9.23 51.65 25

−39.14 −8.21 49.11

5. Molecular Dynamic Simulation Results

Molecular dynamice (“MD”) simulations were carried out using the twodifferent docking poses of compound 12 to determine if there was apreference for one docking pose over another. These data can betterinform which interactions play a role in the results obtained with thecompounds synthesized. The docking results show the higher ranked posewith compound 12 bound into the dinucleotide binding pocket via directH-bond interactions with Ser289 or Arg316 via its hydroxyl moiety(binding mode 1, see FIG. 3 and Table 3). However, there is another posefavorably scored with the morpholine ring of compound 12 interactingwith Ser289 and Arg316 (binding mode 2, see FIG. 3 and Table 3).

MD using the AMBER suite was used to evaluate the energetics of bindingfor both predicted binding modes. Simulations for binding mode 1 showedTr-conjugated electron interactions between compound 12 and Arg 316 aswell as potential for hydrogen bonding between the hydroxyl and Ser289.Analysis of binding mode 2 showed potential π-π interactions betweencompound 12 and Trp756 with more favorable hydrogen bonds with Arg310and Arg316. Further, binding mode 1 is predicted to have hydrogenbonding with Va1590 while binding mode two has van der Waalsinteractions involving the chloro group. MMPBSA analysis of the final100 ps of simulation showed that binding mode 2 was predicted to have afree energy of binding of ˜−40.8 kcal/mol, which is nearly 20 kcal/molmore favorable than ˜−21.0 for binding mode 1. The first 100 ps ofsimulation likely reflect in part the equilibration of the complex suchthat the calculated free energies of binding are not as favorable. Thisfinding contrasts with the rankings of the binding poses during thedocking process. It is possible that this difference arises fromdifferences in the protein structure during docking and MD, with a rigidstructure used to increase the speed of the docking protocol and aflexible structure used for MD.

TABLE 3 Compound No. 12 Compound No. 12 MD (Binding Mode 1) (BindingMode 2) 1-100 ps: −20.2154 −32.9117 ΔG-bind (kcal/mol) 101-200 ps:−21.0263 −40.8046 ΔG-bind (kcal/mol) 150-200 ps: 0.394 1.560 ligand RMSD(Å)

6. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)benzohydrazide

1-(5-Chloro-2-hydroxyphenyl)ethanone (100 mg, 0.586 mmol) andbenzohydrazide (80 mg, 0.586 mmol) were dissolved in methanol (4 mL) inthe presence of acetic acid as a catalyst, and then the reaction mixturewas heated via microwave irradiation to 120° C. for 30 min. Followingcooling, the solvent was removed by vacuum, and the resulting crudematerial was purified by flash column chromatography (2% CH₃OH/CH₂Cl₂)affording the title compound (90 mg) as a solid. ¹H NMR (400 MHz,DMSO-d₆): δ 7.95 (m, 2H), 7.67-7.62 (m, 2H), 7.56 (m, 2H), 7.35 (dd, 1H,J=2.4 & 8.8 Hz), 6.95 (d, 1H, J=8.4 Hz), 3.35 (s, 3H). ESI-MS: 289.0[M+H]⁺.

7. Preparation of(E)-N′-(1-(2,6-dihydroxyphenyl)ethylidene)benzohydrazide

1-(2,6-Dihydroxyphenyl)ethanone (100 mg, 0.657 mmol) and benzohydrazide(89 mg, 0.657 mmol) were dissolved in methanol (4 mL) in the presence ofacetic acid as a catalyst and then the reaction mixture was heated viamicrowave irradiation to 120° C. for 30 min. Following cooling, thesolvent was removed by vacuum, and the resulting crude material waspurified by flash column chromatography (2% CH₃OH/CH₂Cl₂) affording thetitle compound (100 mg) as a solid. ¹H NMR (400 MHz, CD₃OD): δ 7.59 (m,2H), 7.49 (m, 1H), 7.39 (m, 2H), 7.11 (t, 1H, J=8.0 Hz), 6.45 (m, 2H),2.35 (s, 3H). ESI-MS: 271.1 [M+H]⁺.

8. Preparation of 3-(Morpholinosulfonyl)benzoic acid

3-(Chlorosulfonyl)benzoic acid (250 mg, 1.133 mmol) was added tomorpholine (99 mg, 1.133 mmol) in the presence of potassium carbonate(313 mg, 2.266 mmol) in THF (5 mL) at room temperature, and reactionmixture allowed to stirred for 12 h at rt. The reaction was monitored byTLC. After completion of the reaction, the solvent was removed byvacuum, and then compound was purified by column chromatography (3%CH₃OH/CH₂Cl₂) affording the title compound (160 mg) as a solid. ¹H NMR(400 MHz, CD₃OD): δ 8.34 (m, 1H), 8.32 (d, 1H, J=8.0 Hz), 7.99 (m, 1H),7.76 (t, 1H, J=8.0 Hz), 3.70 (m, 4H), 2.98 (m, 4H). ESI-MS: 272.0[M+H]⁺.

9. Preparation of Methyl 3-(morpholinosulfonyl)benzoate

3-(Morpholinosulfonyl)benzoic acid (100 mg, 0.369 mmol) was refluxedovernight in methanol in the presence of catalytic concentrated H₂SO₄ at65° C. The reaction was monitored by TLC. After completion of thereaction, the solvent was removed by vacuum and then compound waspurified by column chromatography to yield the title compound as an offwhite solid (60 mg). ¹H NMR (400 MHz, CDCl₃): δ 8.38 (t, 1H, J=1.6 Hz),8.27 (m, 1H), 7.92 (m, 1H), 7.64 (t, 1H, J=8.0 Hz), 3.95 (s, 3H), 3.73(m, 4H), 3.00 (m, 4H). ESI-MS: 286.1 [M+H]⁺.

10. Preparation of 3-(Morpholinosulfonyl)benzohydrazide

Methyl 3-(morpholinosulfonyl)benzoate (120 mg, 0.421 mmol) was added tohydrazine (17.52 mg, 0.547 mmol) in methanol and refluxed for 12 h at65° C. The reaction was monitored by TLC. Upon completion of thereaction and cooling the reaction mixture, the solvent was removed byvacuum and then compound was purified by column chromatography to yieldthe title compound as an off white solid (90 mg). ¹H NMR (400 MHz,CDCl₃): δ 8.16 (m, 1H), 8.12 (m, 1H), 8.04 (m, 1H), 7.85 (m, 1H), 7.63(t, 1H, J=8.0 Hz), 4.19 (m, 2H), 3.71 (m, 4H), 2.97 (m, 4H). ESI-MS:286.1 [M+H]⁺.

11. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide

1-(5-Chloro-2-hydroxyphenyl)ethanone (20 mg, 0.117 mmol) and3-(morpholinosulfonyl) benzohydrazide (33.5 mg, 0.117 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. Following cooling, the solvent was removed byvacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) affording the title compound (16 mg) asa solid. ¹H NMR (400 MHz, CD₃OD): δ 8.26 (m, 1H), 8.17 (d, 1H, J=8.0Hz), 7.92 (d, 1H, J=8.0 Hz), 7.72 (t, 1H, J=8.0 Hz), 7.48 (d, 1H, J=2.0Hz), 7.22 (m, 1H), 6.91 (d, 1H, J=8.8 Hz), 3.72 (m, 4H), 3.01 (m, 4H),2.43 (s, 3H). ESI-MS: 438.1 [M+H]⁺.

12. Preparation of(E)-N′-(1-(3-chloro-2-fluorophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide

1-(3-Chloro-2-fluorophenyl)ethanone (20 mg, 0.116 mmol) and3-(morpholinosulfonyl) benzohydrazide (33.1 mg, 0.116 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. Following cooling, the solvent was removed byvacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) affording the title compound (22 mg) asa solid. ¹H NMR (400 MHz, CDCl₃): δ 9.43 (s, 1H), 8.37 (m, 1H), 8.16 (m,1H), 7.87 (d, 1H, J=7.2 Hz), 7.65 (m, 1H), 7.41 (m, 1H), 7.10 (t, 1H,J=8.0 Hz), 3.71 (m, 4H), 2.95 (m, 4H), 2.38 (s, 3H). ESI-MS: 440.1[M+H]⁺.

13. Preparation of(E)-N′-(1-(2-chloropyridin-4-yl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide

1-(2-Chloropyridin-4-yl)ethanone (20 mg, 0.129 mmol) and3-(morpholinosulfonyl) benzohydrazide (36.7 mg, 0.129 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. Following cooling, the solvent was removed byvacuum and the resulting crude material was purified by flash columnchromatography affording the title compound in a 60% yield. ¹H NMR (400MHz, CDCl₃): δ 9.43 (m, 1H), 8.39 (m, 2H), 8.15 (d, 1H, J=8.0 Hz), 7.93(d, 1H, J=7.6 Hz), 7.70 (t, 1H, J=7.6 Hz), 7.52 (m, 1H), 3.73 (m, 4H),3.02 (m, 4H), 2.35 (s, 3H). ESI-MS: 423.1 [M+H]⁺.

14. Preparation of(E)-N′-(1-(2,5-dichlorophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide

1-(2,5-Dichlorophenyl)ethanone (20 mg, 0.106 mmol) and3-(morpholinosulfonyl)benzohydrazide (30.2 mg, 0.106 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. Following cooling, the solvent was removed byvacuum and the resulting crude material was purified by flash columnchromatography affording the title compound in a 10 mg yield. ¹H NMR(400 MHz, CDCl₃): δ 8.29 (m, 1H), 8.09 (m, 1H), 7.81 (m, 1H), 7.57 (m,1H), 7.40 (m, 1H), 7.26 (m, 2H), 3.52 (m, 4H), 2.91 (m, 4H), 2.28 (s,3H). ESI-MS: 456.1 [M+H]⁺.

15. Preparation of Methyl 4-hydrazinyl-3-(morpholinosulfonyl)benzoate

Methyl 4-fluoro-3-(morpholinosulfonyl)benzoate (30 mg, 0.099 mmol) wasadded to hydrazine (4.44 mg, 0.138 mmol) in methanol (8 mL) and refluxedfor 5 h at 65° C. The reaction was monitored by TLC. Upon completion ofthe reaction and cooling, the solvent was removed by vacuum, and thecompound was purified by column chromatography affording the titlecompound (20 mg). ¹H NMR (400 MHz, CD₃OD): δ 8.15 (d, 1H, J=2.0 Hz),8.03 (dd, 1H, J=2.4 & 9.2 Hz), 7.48 (d, 1H, J=9.2 Hz), 3.86 (s, 3H),3.67 (m, 4H), 3.04 (m, 4H). ESI-MS: 316.1 [M+H]⁺.

16. Preparation of Methyl 4-fluoro-3-(morpholinosulfonyl)benzoate

4-Fluoro-3-(morpholinosulfonyl)benzoic acid (50 mg, 0.173 mmol) wasrefluxed overnight in the presence of concentrated sulfuric acid (1.117mg, 8.64 μmol) in methanol (8 mL) at 70° C. The reaction was monitoredby TLC. After completion of the reaction, the solvent was removed byvacuum and compound was purified by column chromatography affording thetitle compound (20 mg). ¹H NMR (400 MHz, CD₃OD): δ 8.42 (dd, 1H, J=2.0 &6.4 Hz), 8.33 (m, 1H), 7.49 (t, 1H, J=8.8 Hz), 3.94 (s, 3H), 3.71 (m,4H), 3.16 (m, 4H).

17. Preparation of Methyl 3-bromo-4-chlorobenzoate

3-Bromo-4-chlorobenzoic acid (200 mg, 0.849 mmol) was refluxed in thepresence of concentrated sulfuric acid (5.49 mg, 0.042 mmol) in methanol(10 mL) at 70° C. for overnight. The reaction was monitored by TLC.After completion of the reaction, the solvent was removed by vacuum, andthe compound was purified by column chromatography affording the titlecompound (130 mg). ¹H NMR (400 MHz, CDCl₃): δ 8.29 (d, 1H, J=2.0 Hz),7.91 (dd, 1H, J=2.0 & 8.4 Hz), 7.52 (d, 1H, J=8.4 Hz), 3.92 (s, 3H).ESI-MS: 250.9 [M+H]⁺.

18. Preparation of Methyl 3-(N,N-dimethylsulfamoyl)benzoate

3-(N,N-Dimethylsulfamoyl)benzoic acid (200 mg, 0.872 mmol) was refluxedovernight in the presence of concentrated sulfuric acid (5.64 mg, 0.044mmol) in methanol (10 mL) at 70° C. Reaction was monitored by TLC. Aftercompletion of the reaction, the solvent was removed by vacuum and thencompound was purified by column chromatography affording the titlecompound (125 mg). ¹H NMR (400 MHz, CDCl₃): δ 8.42 (s, 1H), 8.27 (d, 1H,J=8.0 Hz), 7.97 (d, 1H, J=7.2 Hz), 7.65 (t, 1H, J=8.0 Hz), 3.96 (s, 3H),2.74 (s, 6H). ESI-MS: 244.0 [M+H]⁺.

19. Preparation of 3-Bromo-4-chlorobenzohydrazide

Methyl 3-bromo-4-chlorobenzoate (120 mg, 0.481 mmol) was added tohydrazine (23.12 mg, 0.721 mmol) in methanol (8 mL) and refluxed for 12h at 70° C. Reaction was monitored by TLC. After completion of thereaction, the solvent was removed by vacuum and then compound waspurified by column chromatography affording the title compound (30 mg).¹H NMR (400 MHz, CDCl₃): δ 8.02 (d, 1H, J=1.6 Hz), 7.60 (dd, 1H, J=2.0 &8.0 Hz), 7.52 (d, 1H, J=8.0 Hz). ESI-MS: 250.9 [M+H]⁺.

20. Preparation of 3-(Hydrazinecarbonyl)-N,N-dimethylbenzenesulfonamide

Methyl 3-(N,N-dimethylsulfamoyl)benzoate (150 mg, 0.617 mmol) was addedto hydrazine (29.6 mg, 0.925 mmol) in methanol (10 mL) and refluxed for8 h at 65° C. Following cooling, the reaction was monitored by TLC.After completion of the reaction, the solvent was removed by vacuum andthe compound was purified by column chromatography affording the titlecompound (60 mg). ¹H NMR (400 MHz, CDCl₃): δ 8.11 (s, 1H), 8.01 (d, 1H,J=8.4 Hz), 7.92 (d, 1H, J=8.0 Hz), 7.65 (t, 1H, J=8.0 Hz), 2.73 (5, 6H).ESI-MS: 244.0 [M+H]⁺.

21. Preparation of(E)-3-bromo-4-chloro-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)benzohydrazide

3-Bromo-4-chlorobenzohydrazide (30 mg, 0.120 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (20.51 mg, 0.120 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. The reaction was monitored by TLC. Uponcompletion of the reaction and following cooling, the solvent wasremoved by vacuum, and the resulting crude material was purified byflash column chromatography (2% CH₃OH/CH₂Cl₂) affording the titlecompound (15 mg). ¹H NMR (400 MHz, acetone-d₅): δ 8.30 (s, 1H), 7.98 (d,1H, J=8.4 Hz), 7.73 (d, 1H, J=8.4 Hz), 7.61 (d, 1H, J=2.4 Hz), 7.29 (dd,1H, J=2.4 & 8.4 Hz), 6.93 (d, 1H, J=8.8 Hz), 2.55 (s, 3H). ESI-MS: 402.9[M+H]⁺.

22. Preparation of(E)-3-(2-(1-(5-chloro-2-hydroxyphenyl)ethylidene)hydrazinecarbonyl)-N,N-dimethylbenzenesulfonamide

3-(Hydrazinecarbonyl)-N,N-dimethylbenzenesulfonamide (50 mg, 0.0.206mmol) and 1-(5-chloro-2-hydroxyphenyl)ethanone (35.1 mg, 0.206 mmol)were dissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. The reaction was monitored by TLC. Uponcompletion of the reaction and following cooling, the solvent wasremoved by vacuum, and the resulting crude material was purified byflash column chromatography (2% CH₃OH/CH₂Cl₂) affording the titlecompound as a solid (15 mg). ¹H NMR (400 MHz, acetone-d₆): δ 8.29 (m,2H), 8.01 (d, 1H, J=8.4 Hz), 7.83 (t, 1H, J=8.4 Hz), 7.62 (d, 1H, J=2.4Hz), 7.32 (dd, 1H, J=2.4 & 8.8 Hz), 6.96 (d, 1H, J=8.8 Hz), 2.73 (s,6H), 2.58 (s, 3H). ESI-MS: 396.0 [M+H]⁺.

23. Preparation of 5-bromo-6-chloronicotinohydrazide

Methyl 5-bromo-6-chloronicotinate (100 mg, 0.399 mmol) was added tohydrazine (19.19 mg, 0.599 mmol) in methanol (8 mL) and heated overnightat 70° C. The reaction was monitored by TLC. Upon completion of thereaction, the solvent was removed by vacuum, and the compound waspurified by column chromatography affording the title compound (20 mg).¹H NMR (400 MHz, CD₃OD): δ 8.33 (d, 1H, J=2.4 Hz), 8.01 (d, 1H, J=2.4Hz).

24. Preparation of(E)-5-bromo-6-chloro-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)nicotinohydrazide

5-Bromo-6-chloronicotinohydrazide (15 mg, 0.060 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (10.22 mg, 0.060 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. The reaction was monitored by TLC. Uponcompletion of the reaction and following cooling, the solvent wasremoved by vacuum, and the resulting crude material was purified byflash column chromatography (2% CH₃OH/CH₂Cl₂) affording the titlecompound as a solid (8 mg). ¹H NMR (400 MHz, DMSO-d₆): δ 8.39 (d, 1H,J=2.4 Hz), 8.28 (s, 1H), 7.63 (d, 1H, J=2.4 Hz), 7.32 (dd, 1H, J=2.4 &8.8 Hz), 7.06 (d, 1H, J=6.8 Hz), 6.92 (d, 1H, J=9.2 Hz), 6.81 (d, 1H,J=6.8 Hz), 2.47 (s, 3H). ESI-MS: 404.0 [M+H]⁺.

25. Preparation of Methyl 5-chloronicotinate

5-Chloronicotinic acid (200 mg, 1.269 mmol) was refluxed overnight inthe presence of concentrated sulfuric acid (8.20 mg, 0.063 mmol) inmethanol (10 mL) at 70° C. The reaction was monitored by TLC. Aftercompletion of the reaction, the solvent was removed by vacuum and thecompound was purified by column chromatography affording the titlecompound (120 mg). ¹H NMR (400 MHz, CDCl₃): δ 9.07 (d, 1H, J=1.6 Hz),8.72 (d, 1H, J=2.0 Hz), 8.26 (m, 1H), 3.95 (s, 1H).

26. Preparation of Methyl 5-chloronicotinate

5-Chloronicotinic acid (200 mg, 1.269 mmol) was refluxed overnight inthe presence of concentrated sulfuric acid (8.20 mg, 0.063 mmol) inmethanol (8 mL) at 70° C. The reaction was monitored by TLC. Aftercompletion of the reaction, the solvent was removed by vacuum and thecompound was purified by column chromatography affording the titlecompound (120 mg). ¹H NMR (400 MHz, CDCl₃): δ 9.07 (d, 1H, J=1.6 Hz),8.72 (d, 1H, J=2.0 Hz), 8.26 (m, 1H), 3.95 (s, 1H).

27. Preparation of 5-chloronicotinohydrazide

Hydrazine (17.93 mg, 0.560 mmol) was added to methyl 5-chloronicotinate(80 mg, 0.466 mmol) in methanol (8 mL) and heated overnight at 70° C.The reaction was monitored by TLC. After completion of the reaction, thesolvent was removed by vacuum and the compound was purified by columnchromatography affording the title compound (40 mg). ¹H NMR (400 MHz,CD₃OD): δ 8.85 (d, 1H, J=2.0 Hz), 8.70 (d, 1H, J=2.4 Hz), 8.22 (t, 1H,J=2.0 Hz). ESI-MS: 172.0 [M+H]⁺.

28. Preparation of(E)-5-chloro-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)nicotinohydrazide

5-Chloronicotinohydrazide (30 mg, 0.175 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (29.8 mg, 0.175 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. The reaction was monitored by TLC. Uponcompletion of the reaction and following cooling, the solvent wasremoved by vacuum, and the resulting crude material was purified byflash column chromatography (2% CH₃OH/CH₂Cl₂) affording the titlecompound as a solid (20 mg). ¹H NMR (400 MHz, acetone-d₆): δ 9.06 (s,1H), 8.77 (s, 1H), 8.37 (s, 1H), 7.62 (d, 1H, J=2.8 Hz), 7.31 (dd, 1H,J=2.0 & 8.4 Hz), 6.95 (d, 1H, J=8.8 Hz), 2.58 (s, 3H). ESI-MS: 324.0[M+H]⁺.

29. Preparation of(E)-N′-(1-chloro-2-hydroxyphenyl)propylidene)-3-(morpholinosulfonyl)benzohydrazide

3-(Morpholinosulfonyl)benzohydrazide (40 mg, 0.140 mmol) and1-(5-chloro-2-hydroxyphenyl)propan-1-one (25.9 mg, 0.140 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. The reaction was monitored by TLC. Uponcompletion of the reaction and following cooling, the solvent wasremoved by vacuum, and the resulting crude material was purified byflash column chromatography (2% CH₃OH/CH₂Cl₂) affording the titlecompound as a solid (20 mg). ¹H NMR (400 MHz, acetone-d₆): δ 8.26 (m,2H), 8.00 (d, 1H, J=7.6 Hz), 7.84 (t, 1H, J=8.0 Hz), 7.64 (d, 1H, J=2.4Hz), 7.33 (m, 1H), 6.98 (d, 1H, J=9.2 Hz), 3.69 (m, 4H), 3.10 (q, 2H,J=7.6 Hz), 2.99 (m, 4H), 1.26 (t, 3H, J=7.6 Hz). ESI-MS: 452.1 [M+H]⁺.

30. Preparation of(E)-3-(morpholinosulfonyl)-N′-(1-(pyridin-3-yl)ethylidene)benzohydrazide

3-(Morpholinosulfonyl)benzohydrazide (40 mg, 0.140 mmol) and1-(pyridin-3-yl)ethanone (16.98 mg, 0.140 mmol) were dissolved inmethanol (4 mL) in the presence of acetic acid as a catalyst, and thereaction mixture was heated via microwave irradiation to 120° C. for 30min. The reaction was monitored by TLC. Upon completion of the reactionand following cooling, the solvent was removed by vacuum, and theresulting crude material was purified by flash column chromatography (2%CH₃OH/CH₂Cl₂) affording the title compound as a solid (15 mg). ¹H NMR(400 MHz, CDCl₃): δ 9.53 (bs, 1H), 8.87 (s, 1H), 8.59 (m, 1H), 8.39 (m,1H), 8.17 (m, 1H), 7.98 (m, 1H), 7.89 (d, 1H, J=8.0 Hz), 7.67 (t, 1H,J=8.0 Hz), 7.32 (m, 1H), 3.70 (m, 4H), 3.00 (m, 4H), 2.39 (s, 3H).ESI-MS: 389.0 [M+H]⁺.

31. Preparation of 3-morpholinobenzohydrazide

Methyl 3-morpholinobenzoate (100 mg, 0.452 mmol) was added to hydrazine(14.48 mg, 0.452 mmol) in methanol (10 mL) and refluxed for 12 h at 65°C. The reaction was monitored by TLC. Upon completion of the reactionand following cooling, the solvent was removed by vacuum and thecompound was purified by column chromatography (2% CH3OH/CH₂Cl₂)affording the title compound as a solid (52 mg). ¹H NMR (400 MHz,DMSO-d₆): δ 9.69 (s, 1H), 7.35 (s, 1H), 7.27 (m, 2H), 7.07 (m, 1H), 4.45(bs, 2H), 3.74 (m, 4H), 3.14 (m, 4H). ESI-MS: 222.1 [M+H]⁺.

32. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-morpholinobenzohydrazide

1-(5-Chloro-2-hydroxyphenyl)ethanone (40 mg, 0.234 mmol) and3-morpholinobenzohydrazide (51.9 mg, 0.234 mmol) were dissolved inmethanol (4 mL) in the presence of acetic acid as a catalyst, and thereaction mixture was heated via microwave irradiation to 120° C. for 30min. The reaction was monitored by TLC. Upon completion of the reactionand following cooling, the solvent was removed by vacuum, and theresulting crude material was purified by flash column chromatography (2%CH₃OH/CH₂Cl₂) affording the title compound (60 mg) as a solid. ¹H NMR(400 MHz, DMSO-d₆): δ 7.65 (d, 1H, J=2.4 Hz), 7.42-7.32 (m, 4H), 7.20(m, 1H), 6.94 (d, 1H, J=8.8 Hz), 3.77 (m, 4H), 3.19 (m, 4H), 2.48 (s,3H). ESI-MS: 374.1 [M+H]⁺.

33. Preparation of 5-(Methylsulfonyl)nicotinohydrazide

Methyl 5-(methylsulfonyl)nicotinate (100 mg, 0.465 mmol) were added tohydrazine (17.87 mg, 0.558 mmol) in methanol (10 mL) and refluxed for 12h at 70° C. The reaction was monitored by TLC. Upon completion of thereaction and following cooling, the solvent was removed by vacuum andthe compound was purified by flash column chromatography (3%CH₃OH/CH₂Cl₂) afforded the title compound (83 mg, 80% yield) as a solid.¹H NMR (400 MHz, CDCl₃): δ 9.20 (d, 1H, J=2.0 Hz), 9.17 (d, 1H, J=2.0Hz), 8.61 (s, 1H), 3.11 (s, 3H). ESI-MS: 216.1 [M+H]⁺.

34. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-5-(methylsulfonyl)nicotinohydrazide

1-(5-Chloro-2-hydroxyphenyl)ethanone (50 mg, 0.293 mmol) and5-(methylsulfonyl) nicotinohydrazide (63.1 mg, 0.293 mmol) weredissolved in methanol (4 mL) in the presence of acetic acid as acatalyst, and the reaction mixture was heated via microwave irradiationto 120° C. for 30 min. The reaction was monitored by TLC. Uponcompletion of the reaction and following cooling, the solvent wasremoved by vacuum, and the resulting crude material was purified byflash column chromatography (3% CH₃OH/CH₂Cl₂) affording the titlecompound (70 mg, 63.0% yield) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ11.86 (s, 1H), 9.37 (s, 1H), 9.27 (s, 1H), 8.76 (s, 1H), 7.68 (s, 1H),7.36 (d, 1H, J=8.8 Hz), 6.97 (d, 1H, J=8.8 Hz), 3.42 (s, 3H), 2.53 (s,3H). ESI-MS: 368.8 [M+H]⁺.

35. Preparation of 3-(Methylsulfonyl)benzohydrazide

Methyl 3-(methylsulfonyl)benzoate (100 mg, 0.467 mmol) was added tohydrazine (22.44 mg, 0.700 mmol)) in methanol (10 mL) and refluxed for12 h at 70° C. The reaction was monitored by TLC. Upon completion of thereaction and following cooling, the solvent was removed by vacuum, andthe compound was purified by flash column chromatography (3%CH₃OH/CH₂Cl₂) affording the title compound (80 mg, 80% yield) as asolid. ¹H NMR (400 MHz, CDCl₃): δ 8.28 (s, 1H), 8.07 (d, 1H, J=7.6 Hz),8.01 (d, 1H, J=7.6 Hz), 7.62 (t, 1H, J=7.6 Hz), 3.04 (5, 3H). ESI-MS:215.1 [M+H]⁺.

36. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(methylsulfonyl)benzohydrazide

1-(5-Chloro-2-hydroxyphenyl)ethanone (55 mg, 0.322 mmol) and3-(methylsulfonyl)benzohydrazide (69.1 mg, 0.322 mmol) were dissolved inmethanol (5 mL) in the presence of acetic acid as a catalyst, and thereaction mixture was heated via microwave irradiation to 120° C. for 30min. The reaction was monitored by TLC. Upon completion of the reactionand following cooling, the solvent was removed by vacuum, and theresulting crude material was purified by flash column chromatography (3%CH₃OH/CH₂Cl₂) affording the title compound (75 mg, 63.4% yield) as asolid. ¹H NMR (400 MHz, CD₃OD): δ 8.49 (s, 1H), 8.26 (d, 1H, J=8.4 Hz),8.18 (d, 1H, J=7.6 Hz), 7.80 (t, 1H, J=7.6 Hz), 7.60 (d, 1H, J=2.4 Hz),7.27 (m, 1H), 6.93 (d, 1H, J=8.8 Hz), 3.19 (s, 3H), 2.49 (s, 3H).ESI-MS: 367.8 [M+H]⁺.

37. Preparation of 3-((4-methylpiperidin-1-yl)sulfonyl)benzoic acid

4-methylpiperidine (180 mg, 1.813 mmol) was added to the3-(chlorosulfonyl)benzoic acid (200 mg, 0.906 mmol) in presence ofPotassiumcarbonate (251 mg, 1.813 mmol) in THF (Volume: 5 ml) at roomtemperature and the reaction mixture was stirred for 12 h at roomtemperature. Reaction was monitored by TLC. After completion of thereaction, the solvent was removed by vacuum and then compound waspurified by column chromatography (3% CH₃OH/CH₂Cl₂) afforded the titlecompound as a solid. 1H NMR (CD₃OD, 400 MHz): δ 8.32 (m, 1H), 8.27 (m,1H), 7.96 (m, 1H), 7.72 (t, 1H, J=8.0 Hz), 3.72 (m, 2H), 2.27 (m, 2H),1.68 (m, 2H), 1.29 (m, 1H), 1.21 (m, 2H), 0.88 (d, 3H, J=6.4 Hz).ESI-MS: 284.1 [M+H]+

38. Preparation of methyl 3-((4-methylpiperidin-1-yl)sulfonyl)benzoate

3-((4-methylpiperidin-1-yl)sulfonyl)benzoic acid (120 mg, 0.424 mmol)was refluxed in the presence of con. Sulfuric acid (2.74 mg, 0.021 mmol)in methanol at 70° C. for overnight. Reaction was monitored by TLC.After completion of the reaction, the solvent was removed by vacuum andthen compound was purified by flash chromatography afforded the methyl3-((4-methylpiperidin-1-yl)sulfonyl)benzoate (100 mg, 0.319 mmol, 75%yield). 1H NMR (CDCl3, 400 MHz): δ 8.39 (m, 1H), 8.25 (m, 1H), 7.94 (m,1H), 7.62 (t, 1H, J=7.6 Hz), 3.95 (s, 3H), 3.77 (m, 2H), 2.25 (m, 2H),1.67 (m, 2H), 1.29 (m, 3H), 0.90 (d, 3H, J=4.8 Hz). ESI-MS: 298.1[M+H]+

39. Preparation of 2-(morpholinosulfonyl)benzohydrazide

Hydrazine (22.46 mg, 0.701 mmol) was added to the methyl2-(morpholinosulfonyl)benzoate (100 mg, 0.350 mmol) in methanol andrefluxed for 12 h at 70° C. Following cooling, Reaction was monitored byTLC. After completion of the reaction, the solvent was removed by vacuumand then compound was purified by flash chromatography afforded thetitle compound 2-(morpholinosulfonyl)benzohydrazide (40 mg, 0.129 mmol,36.8% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ 7.86 (m, 1H),7.66-7.56 (m, 2H), 7.52 (dd, 1H, J=1.2 & 7.6 Hz), 7.40 (m, 1H), 4.09 (m,2H), 3.70 (m, 4H), 3.15 (m, 4H). ESI-MS: 286.1[M+H]+

40. Preparation of 3-((4-methylpiperidin-1-yl)sulfonyl)benzohydrazide

Methyl 3-((4-methylpiperidin-1-yl)sulfonyl)benzoate (100 mg, 0.336 mmol)was added to the hydrazine (21.55 mg, 0.673 mmol) in methanol andrefluxed for 8 h at 65° C. Following cooling, reaction was monitored byTLC. After completion of the reaction, the solvent was removed by vacuumand then compound was purified by column chromatography to yield3-((4-methylpiperidin-1-yl)sulfonyl)benzohydrazide (70 mg, 0.217 mmol,64.4% yield). 1H NMR (CD3OD, 400 MHz): δ 8.16 (m, 1H), 8.05 (m, 1H),7.91 (m, 1H), 7.70 (t, 1H, J=7.6 Hz), 3.74 (m, 2H), 2.28 (m, 2H), 1.69(m, 2H), 1.32-1.16 (m, 3H), 0.90 (d, 3H, J=6.0 Hz). ESI-MS: 298.1[M+H]+

41. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-((4-methylpiperidin-1-yl)sulfonyl)benzohydrazide

3-((4-methylpiperidin-1-yl)sulfonyl)benzohydrazide (70 mg, 0.235 mmol)and 1-(5-chloro-2-hydroxyphenyl)ethanone (40.2 mg, 0.235 mmol) wasdissolved in Methanol (Volume: 4 ml) in the presence of acetic acid as acatalyst and then the reaction mixture was heated via microwaveirradiation to 120° C. for 30 min. Reaction was monitored by TLC. Aftercompletion of the reaction, following cooling, the solvent was removedby vacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-((4-methylpiperidin-1-yl)sulfonyl)benzohydrazide(15 mg, 0.032 mmol, 13.60% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ8.11 (m, 2H), 7.81 (m, 1H), 7.59 (m, 1H), 7.39 (m, 1H), 7.19 (m, 1H),6.89 (m, 1H), 3.69 (m, 2H), 2.41 (m, 2H), 2.24 (m, 2H), 1.63 (m, 2H),1.24 (m, 4H), 0.87 (d, 3H, J=4.4 Hz). Mass [M+H]+: 450.2

42. Preparation of(E)-N′-(1-(5-chloro-2-fluorophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide

1-(5-chloro-2-fluorophenyl)ethanone (20 mg, 0.116 mmol) and3-(morpholinosulfonyl)benzohydrazide (33.1 mg, 0.116 mmol) was dissolvedin Methanol (Volume: 4 ml) in the presence of acetic acid as a catalystand then the reaction mixture was heated via microwave irradiation to120° C. for 30 min. Reaction was monitored by TLC. After completion ofthe reaction, following cooling, the solvent was removed by vacuum andthe resulting crude material was purified by flash column chromatography(2% CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-fluorophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide(10 mg, 0.022 mmol, 19.22% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ8.26 (m, 1H), 8.09 (m, 1H), 7.80 (d, 1H, J=7.6 Hz), 7.58 (t, 1H, J=7.6Hz), 7.37 (m, 1H), 7.21 (m, 1H), 6.95 (m, 1H), 3.61 (m, 4H), 2.90 (m,4H), 2.29 (s, 3H). Mass [M+H]+: 440.1

43. Preparation of methyl 3-(pyrrolidin-1-ylsulfonyl)benzoate

3-(pyrrolidin-1-ylsulfonyl)benzoic acid (200 mg, 0.783 mmol) wasrefluxed in the presence of con. Sulfuric acid (5.06 mg, 0.039 mmol) inmethanol at 70° C. for overnight. Reaction was monitored by TLC. Aftercompletion of the reaction, solvent was removed by vacuum and thencompound was purified by flash chromatography afforded the methyl3-(pyrrolidin-1-ylsulfonyl)benzoate (150 mg, 0.535 mmol, 68.3% yield).1H NMR (CDCl3, 400 MHz): δ 8.47 (m, 1H), 8.25 (d, 1H, J=7.6 Hz), 8.02(dt, 1H, J=1.2 & 8.0 Hz), 7.63 (t, 1H, J=7.6 Hz), 3.96 (s, 3H), 3.27 (m,4H), 1.77 (m, 4H). Mass [M+H]+: 270.1

44. Preparation of methyl 3-(N-methylsulfamoyl)benzoate

3-(N-methylsulfamoyl)benzoic acid (200 mg, 0.929 mmol) was refluxed inthe presence of concentrated sulfuric acid (6.01 mg, 0.046 mmol) inmethanol at 70° C. for overnight. Reaction was monitored by TLC. Aftercompletion of the reaction, the solvent was removed by vacuum and thencompound was purified by flash chromatography afforded the methyl3-(N-methylsulfamoyl)benzoate (120 mg, 0.497 mmol, 53.5% yield). 1H NMR(CDCl3, 400 MHz): δ 8.51 (m, 1H), 8.25 (m, 1H), 8.06 (dt, 1H, J=1.2 &8.0 Hz), 7.63 (t, 1H, J=7.6 Hz), 3.96 (s, 3H), 2.69 (s, 3H). Mass[M+H]+: 230.1

45. Preparation of 3-(pyrrolidin-1-ylsulfonyl)benzohydrazide

Methyl 3-(pyrrolidin-1-ylsulfonyl)benzoate (150 mg, 0.557 mmol) wasadded to the hydrazine (35.7 mg, 1.114 mmol) in methanol and refluxedfor 12 h at 65° C. Following cooling, reaction was monitored by TLC.After completion of the reaction, the solvent was removed by vacuum andthen compound was purified by column chromatography to yield3-(pyrrolidin-1-ylsulfonyl)benzohydrazide (110 mg, 0.396 mmol, 71.1%yield). 1H NMR (CDCl3, 400 MHz): δ 8.18 (m, 1H), 8.03 (d, 1H, J=7.6 Hz),7.97 (d, 1H, J=8.0 Hz), 7.78 (bs, 1H), 7.63 (t, 1H, J=7.6 Hz), 4.17 (bs,2H), 3.25 (m, 4H), 1.77 (m, 4H). Mass [M+H]+: 270.1

46. Preparation of 3-(hydrazinecarbonyl)-N-methylbenzenesulfonamide

Hydrazine (43.3 mg, 1.352 mmol) was added to the methyl3-(N-methylsulfamoyl)benzoate (155 mg, 0.676 mmol) in methanol andrefluxed for 12 h at 65° C. Following cooling, reaction was monitored byTLC. After completion of the reaction, the solvent was removed by vacuumand then compound was purified by column chromatography to yield3-(hydrazinecarbonyl)-N-methylbenzenesulfonamide (120 mg, 0.502 mmol,74.3% yield). 1H NMR (CDCl3, 400 MHz): δ 8.25 (m, 1H), 8.01 (m, 2H),7.64 (m, 2H), 4.63 (m, 1H), 4.17 (m, 2H), 2.69 (d, 3H, J=5.2 Hz).ESI-MS: 230.0 [M+H]+

47. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-2-(morpholinosulfonyl)benzohydrazide

2-(morpholinosulfonyl)benzohydrazide (30 mg, 0.105 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (17.94 mg, 0.105 mmol) wasdissolved in Methanol (Volume: 4 ml) in the presence of acetic acid as acatalyst and then the reaction mixture was heated via microwaveirradiation to 120° C. for 30 min. Reaction was monitored by TLC. Aftercompletion of the reaction, following cooling, the solvent was removedby vacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-2-(morpholinosulfonyl)benzohydrazide(10 mg, 0.022 mmol, 21.28% yield) as a solid. 1H NMR (CD₃OD, 400 MHz): δ7.95 (d, 1H, J=8.0 Hz), 7.95-7.70 (m, 2H), 7.66 (d, 1H, J=7.6 Hz), 7.56(d, 1H, J=2.8 Hz), 7.25 (dd, 1H, J=2.8 & 8.8 Hz), 6.91 (d, 1H, J=8.4Hz), 3.66 (m, 4H), 3.2 (m, 4H), 2.36 (s, 3H). Mass [M+H]+: 438.1

48. Preparation of(E)-3-(2-(1-(5-chloro-2-hydroxyphenyl)ethylidene)hydrazinecarbonyl)-N-methylbenzenesulfonamide

3-(hydrazinecarbonyl)-N-methylbenzenesulfonamide (120 mg, 0.523 mmol)and 1-(5-chloro-2-hydroxyphenyl)ethanone (89 mg, 0.523 mmol) wasdissolved in Methanol (Volume: 4 ml) in the presence of acetic acid as acatalyst and then the reaction mixture was heated via microwaveirradiation to 120° C. for 30 min. Reaction was monitored by TLC. Aftercompletion of the reaction, following cooling, the solvent was removedby vacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) afforded the title compound(E)-3-(2-(1-(5-chloro-2-hydroxyphenyl)ethylidene)hydrazinecarbonyl)-N-methylbenzenesulfonamide(75 mg, 0.192 mmol, 36.8% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ8.21 (m, 1H), 8.06 (m, 1H), 7.95 (d, 1H, J=7.6 Hz), 7.59 (t, 1H, J=8.0Hz), 7.39 (d, 1H, J=2.4 Hz), 7.18 (m, 1H), 6.90 (d, 1H, J=8.0 Hz), 2.56(s, 3H), 2.36 (5, 3H). Mass [M+H]+: 382.1

49. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(pyrrolidin-1-ylsulfonyl)benzohydrazide

3-(pyrrolidin-1-ylsulfonyl)benzohydrazide (105 mg, 0.390 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (66.5 mg, 0.390 mmol) was dissolvedin Methanol (Volume: 4 ml) in the presence of acetic acid as a catalystand then the reaction mixture was heated via microwave irradiation to120° C. for 30 min. Reaction was monitored by TLC. After completion ofthe reaction, following cooling, the solvent was removed by vacuum andthe resulting crude material was purified by flash column chromatography(2% CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(pyrrolidin-1-ylsulfonyl)benzohydrazide(70 mg, 0.163 mmol, 41.7% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ8.18 (m, 1H), 8.13 (m, 1H), 7.95 (d, 1H, J=7.6 Hz), 7.65 (t, 1H, J=7.6Hz), 7.41 (m, 1H), 7.21 (m, 1H), 6.93 (d, 1H, J=8.8 Hz), 3.23 (m, 4H),2.39 (s, 3H), 1.75 (m, 4H). Mass [M+H]+: 422.1

50. Preparation of methyl 3-(1,1-dioxidothiomorpholino)benzoate

3-(1,1-dioxidothiomorpholino)benzoic acid (100 mg, 0.392 mmol) wasrefluxed in the presence of con. Sulfuric acid (2.53 mg, 0.020 mmol) inmethanol(5 mL) at 70° C. for overnight. Reaction was monitored by TLC.After completion of the reaction, the solvent was removed by vacuum andthen compound was purified by flash chromatography afforded the methyl3-(1,1-dioxidothiomorpholino)benzoate (99 mg, 0.353 mmol, 90% yield). 1HNMR (CDCl3, 400 MHz): δ 7.58 (m, 2H), 7.36 (t, 1H, J=8.0 Hz), 7.09 (m,1H), 3.91 (s, 3H), 3.89 (m, 4H), 3.11 (m, 4H). Mass [M+H]+: 270.1

51. Preparation of 3-(1,1-dioxidothiomorpholino)benzohydrazide

Methyl 3-(1,1-dioxidothiomorpholino)benzoate (95 mg, 0.353 mmol) wasadded to the hydrazine (22.61 mg, 0.705 mmol) in methanol and refluxedfor 12 h at 65° C. Following cooling, Reaction was monitored by TLC.After completion of the reaction, the solvent was removed by vacuum andthen compound was purified by column chromatography (2% CH3OH/CH2Cl2)afforded the title compound 3-(1,1-dioxidothiomorpholino)benzohydrazide(32 mg, 0.109 mmol, 31.0% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ7.34 (m, 1H), 7.29 (t, 1H, J=8.4 Hz), 7.18 (d, 1H, J=7.6 Hz), 6.70 (dd,1H, J=4.8 & 8.0 Hz), 3.85 (m, 4H), 3.05 (m, 4H). Mass [M+H]+: 270.1

52. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(1,1-dioxidothiomorpholino)benzohydrazide

3-(1,1-dioxidothiomorpholino)benzohydrazide (30 mg, 0.111 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (19.00 mg, 0.111 mmol) wasdissolved in methanol (Volume: 4 ml) in the presence of acetic acid as acatalyst and then the reaction mixture was heated via microwaveirradiation to 120° C. for 30 min. Reaction was monitored by TLC. Aftercompletion of the reaction, following cooling, the solvent was removedby vacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(1,1-dioxidothiomorpholino)benzohydrazide(15 mg, 0.035 mmol, 31.3% yield) as a solid. 1H NMR (DMSO-d6, 400 MHz):δ 7.65 (d, 1H, J=2.0 Hz), 7.47 (m, 1H), 7.41 (t, 1H, J=7.6 Hz),7.36-7.27 (m, 3H), 6.94 (d, 1H, J=8.8 Hz), 3.87 (m, 4H), 3.17 (m, 4H),2.48 (s, 3H). Mass [M+H]+: 422.2

53. Preparation of(E)-N′-(1-(5-chloro-2-nitrophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide

1-(5-chloro-2-nitrophenyl)ethanone (30 mg, 0.150 mmol) and3-(morpholinosulfonyl)benzohydrazide (42.9 mg, 0.150 mmol) was dissolvedin Methanol (Volume: 4 ml) in the presence of acetic acid as a catalystand then the reaction mixture was heated via microwave irradiation to120° C. for 30 min. Reaction was monitored by TLC. After completion ofthe reaction, following cooling, the solvent was removed by vacuum andthe resulting crude material was purified by flash column chromatography(2% CH₃OH/CH₂Cl₂), afforded the product(E)-N′-(1-(5-chloro-2-nitrophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide(15 mg, 0.030 mmol, 20.09% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ8.20 (m, 1H), 8.07 (m, 1H), 7.88 (m, 1H), 7.66 (m, 1H), 7.51 (m, 2H),7.39 (m, 1H), 3.69 (m, 4H), 2.99 (m, 4H), 2.29 (s, 3H). Mass [M+H]+:468.0

54. Preparation of methyl 3-sulfamoylbenzoate

3-sulfamoylbenzoic acid (150 mg, 0.746 mmol) was refluxed in thepresence of concentrated Sulfuric acid (4.82 mg, 0.037 mmol) in methanol(5 mL) at 70° C. for overnight. Reaction was monitored by TLC. Aftercompletion of the reaction, Solvent was removed by vacuum and thencompound was purified by flash chromatography afforded the methyl3-sulfamoylbenzoate (115 mg, 0.524 mmol, 70.2% yield) as a solid. 1H NMR(CDCl3, 400 MHz): δ 8.53 (m, 1H), 8.18 (d, 1H, J=8.0 Hz), 8.08 (d, 1H,J=7.6 Hz), 7.57 (t, 1H, J=8.0 Hz), 3.92 (s, 3H). Mass [M+H]+: 216.0

55. Preparation of methyl 4-(morpholinosulfonyl)benzoate

4-(morpholinosulfonyl)benzoic acid (150 mg, 0.553 mmol) was refluxed inthe presence of con. Sulfuric acid (3.57 mg, 0.028 mmol) in methanol at70° C. for overnight. Reaction was monitored by TLC. After completion ofthe reaction, solvent was removed by vacuum and then compound waspurified by flash chromatography afforded the methyl4-(morpholinosulfonyl)benzoate (135 mg, 0.464 mmol, 84% yield). 1H NMR(CDCl3, 400 MHz): δ 8.21 (m, 2H), 7.82 (m, 2H), 3.97 (s, 3H), 3.4 (m,4H)_(,) 3.02 (m, 4H). Mass [M+H]+: 286.0

56. Preparation of 3-(hydrazinecarbonyl)benzenesulfonamide

Methyl 3-sulfamoylbenzoate (110 mg, 0.511 mmol) was added to thehydrazine (32.8 mg, 1.022 mmol) in methanol and refluxed for 8 h at 65°G. Following cooling, reaction was monitored by TLC. After completion ofthe reaction, the solvent was removed by vacuum and then compound waspurified by flash chromatography (5% methanol/DCM) to yield the3-(hydrazinecarbonyl)benzenesulfonamide (57 mg, 0.260 mmol, 50.8% yield)as a white solid. 1H NMR (CD3OD, 400 MHz): δ 8.32 (m, 1H), 8.04 (d, 1H,J=7.6 Hz), 7.97 (d, 1H, J=7.6 Hz), 7.63 (t, 1H, J=8.0 Hz). Mass [M+H]+:216.0

57. Preparation of 4-(morpholinosulfonyl)benzohydrazide

Methyl 4-(morpholinosulfonyl)benzoate (135 mg, 0.473 mmol) was added tothe hydrazine (30.3 mg, 0.946 mmol) in methanol and refluxed for 8 h at65° C. Following cooling, reaction was monitored by TLC. Aftercompletion of the reaction, the solvent was removed by vacuum and thencompound was purified by flash chromatography (3% methanol/DCM) to yieldthe 4-(morpholinosulfonyl)benzohydrazide (102 mg, 0.350 mmol, 74.0%yield) as a white solid. 1H NMR (CDCl3, 400 MHz): δ 7.94 (m, 2H), 7.79(m, 2H), 3.72 (m, 4H), 2.99 (m, 4H). Mass [M+H]+: 286.0

58. Preparation of(E)-3-(2-(1-(5-chloro-2-hydroxyphenyl)ethylidene)hydrazinecarbonyl)benzenesulfonamide

3-(hydrazinecarbonyl)benzenesulfonamide (50 mg, 0.232 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (39.6 mg, 0.232 mmol) was dissolvedin methanol (Volume: 4 ml) in the presence of acetic acid as a catalystand then the reaction mixture was heated via microwave irradiation to120° C. for 30 min. Reaction was monitored by TLC. After completion ofthe reaction, following cooling, the solvent was removed by vacuum andthe resulting crude material was purified by flash column chromatography(2% CH₃OH/CH₂Cl₂) afforded the product(E)-3-(2-(1-(5-chloro-2-hydroxyphenyl)ethylidene)hydrazinecarbonyl)benzenesulfonamide (36 mg, 0.094 mmol,40.4% yield) as a solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.34 (s, 1H), 8.15(d, 1H, J=7.6 Hz), 8.02 (d, 1H, J=7.6 Hz), 7.73 (t, 1H, J=8.0 Hz), 7.64(m, 1H), 7.51 (bs, 2H), 7.32 (dd, 1H, J=2.4 & 8.4 Hz), 6.92 (d, 1H,J=8.4 Hz), 2.49 (s, 3H). Mass [M+H]+: 368.0

59. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-4-(morpholinosulfonyl)benzohydrazide

4-(morpholinosulfonyl)benzohydrazide (100 mg, 0.350 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (59.8 mg, 0.350 mmol) was dissolvedin methanol (Volume: 4 ml) in the presence of acetic acid as a catalystand then the reaction mixture was heated via microwave irradiation to120° C. for 30 min. Reaction was monitored by TLC. After completion ofthe reaction, following cooling, the solvent was removed by vacuum andthe resulting crude material was purified by flash column chromatography(2% CH₃OH/CH₂Cl₂) afforded the product(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-4-(morpholinosulfonyl)benzohydrazide (80 mg, 0.177 mmol,50.6% yield) as a solid. 1H NMR (DMSO-d6, 400 MHz): δ 8.16 (m, 2H), 7.89(m, 2H), 7.67 (d, 1H, J=2.4 Hz), 7.35 (dd, 1H, J=2.4 & 8.8 Hz), 6.95 (d,1H, J=8.4 Hz), 3.64 (m, 4H), 2.92 (m, 4H), 2.49 (s, 3H). Mass [M+H]+:438.0

60. Preparation of 3-((4-methylpiperazin-1-yl)sulfonyl)benzoic acid

3-(chlorosulfonyl)benzoic acid (200 mg, 0.906 mmol) was added to the1-methylpiperazine (100 mg, 0.997 mmol) in presence of potassiumcarbonate (251 mg, 1.813 mmol) in THF (Volume: 5 ml) at room temperatureand the reaction mixture was stirred for 12 h at room temperature.Reaction was monitored by TLC. After completion of the reaction, thesolvent was removed by vacuum and then compound was purified by columnchromatography (3% CH₃OH/CH₂Cl₂) afforded the product3-((4-methylpiperazin-1-yl)sulfonyl)benzoic acid (100 mg, 0.320 mmol,35.3% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ 7.77 (m, 2H),7.63-7.55 (m, 2H), 3.04 (m, 4H), 2.46 (m 4H), 2.31 (s, 3H). Mass [M+H]+:285.1

61. Preparation of methyl 3-((4-methylpiperazin-1-yl)sulfonyl)benzoate

3-((4-methylpiperazin-1-yl)sulfonyl)benzoic acid (250 mg, 0.879 mmol)was refluxed in the presence of concentrated sulfuric acid (5.68 mg,0.044 mmol) in methanol at 70° C. for overnight. Reaction was monitoredby TLC. After completion of the reaction, solvent was removed by vacuumand the crude material was used for further reaction withoutpurification.

62. Preparation of 3-((4-methylpiperazin-1-yl)sulfonyl)benzohydrazide

Methyl 3-((4-methylpiperazin-1-yl)sulfonyl)benzoate (200 mg, 0.670 mmol)was added to the hydrazine (43.0 mg, 1.341 mmol) in methanol andrefluxed for 8 h at 65° C. Following cooling, reaction was monitored byTLC. After completion of the reaction, the solvent was removed by vacuumand then compound was purified by flash chromatography (3% methanol/DCM)to yield the 3-((4-methylpiperazin-1-yl)sulfonyl)benzohydrazide (125 mg,0.406 mmol, 60.6% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz): δ10.08 (s, 1H), 8.12 (m, 2H), 7.84 (d, 1H, J=7.6 Hz), 7.72 (t, 1H, J=7.6Hz), 4.57 (m, 1H), 2.88 (m, 4H), 2.32 (m, 4H), 2.10 (s, 3H). Mass[M+H]+: 298.9

63. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-((4-methylpiperazin-1-yl)sulfonyl)benzohydrazide

3-((4-methylpiperazin-1-yl)sulfonyl)benzohydrazide (85 mg, 0.285 mmol)and 1-(5-chloro-2-hydroxyphenyl)ethanone (48.6 mg, 0.285 mmol) wasdissolved in methanol (Volume: 4 ml) in the presence of acetic acid as acatalyst and then the reaction mixture was heated via microwaveirradiation to 120° C. for 30 min. Reaction was monitored by TLC. Aftercompletion of the reaction, following cooling, the solvent was removedby vacuum and the resulting crude material was purified by flash columnchromatography (2% CH₃OH/CH₂Cl₂) afforded the product(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-((4-methylpiperazin-1-yl)sulfonyl)benzohydrazide(70 mg, 0.152 mmol, 53.4% yield) as a solid. 1H NMR (CD3OD, 400 MHz): δ8.29 (s, 1H), 8.21 (d, 1H, J=7.2 Hz), 7.99 (d, 1H, J=8.0 Hz), 7.78 (t,1H, J=7.6 Hz), 7.59 (d, 1H, J=2.4 Hz), 7.27 (dd, 1H, J=2.4 & 9.2 Hz),6.92 (d, 1H, J=8.8 Hz), 3.09 (m, 4H), 2.54 (m, 4H), 2.48 (s, 3H), 2.28(s, 3H). Mass [M+H]+: 450.9

64. Preparation of 3-(piperidin-1-ylsulfonyl)benzohydrazide

Methyl 3-(piperidin-1-ylsulfonyl)benzoate (150 mg, 0.529 mmol) was addedto the hydrazine (50.9 mg, 1.588 mmol) in methanol and refluxed for 8 hat 65° C. Following cooling, reaction was monitored by TLC. Aftercompletion of the reaction, solvent was removed by vacuum and thencompound was purified by flash chromatography (3% methanol/DCM) to yieldthe 3-(piperidin-1-ylsulfonyl)benzohydrazide (70 mg, 0.245 mmol, 46.2%yield) as a white solid. 1H NMR (CD3OD, 400 MHz): δ 8.17 (t, 1H, J=1.2Hz), 8.05 (dt, 1H, J=1.2 & 8.0 Hz), 7.90 (dt, 1H, J=1.2 & 8.0 Hz), 7.69(t, 1H, J=7.6 Hz), 2.99 (m, 4H), 1.62 (m, 4H), 1.43 (m, 2H). Mass[M+H]+: 284.1

65. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(piperidin-1-ylsulfonyl)benzohydrazide

3-(piperidin-1-ylsulfonyl)benzohydrazide (65 mg, 0.229 mmol) and1-(5-chloro-2-hydroxyphenyl)ethanone (39.1 mg, 0.229 mmol) was dissolvedin methanol (Volume: 4 ml) in the presence of acetic acid as a catalystand then the reaction mixture was heated via microwave irradiation to120° C. for 30 min. Reaction was monitored by TLC. After completion ofthe reaction, following cooling, the solvent was removed by vacuum andthe resulting crude material was purified by flash column chromatography(2% CH₃OH/CH₂Cl₂) afforded the product(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(piperidin-1-ylsulfonyl)benzohydrazide(55 mg, 0.124 mmol, 53.9% yield) as a solid. 1H NMR (CDCl3, 400 MHz): δ8.09 (m, 2H), 7.85 (d, 1H, J=8.0 Hz), 7.62 (t, 1H, J=8.0 Hz), 7.41 (d,1H, J=2.4 Hz), 7.22 (d, 1H, J=8.0 Hz), 6.93 (d, 1H, J=8.8 Hz), 2.97 (m,4H), 2.41 (s, 3H), 1.61 (m, 4H), 1.40 (m, 2H). Mass [M+H]+: 436.9

66. Preparation of 4-chloro-2-(4-methyl-1H-pyrazol-5-yl)phenol

A mixture of (E)-3-(5-chloro-2-hydroxyphenyl)-2-methylacrylaldehyde (40mg, 0.203 mmol) and 4-methylbenzenesulfonohydrazide (41.7 mg, 0.224mmol) in Acetonitrile (3 mL) were stirred at room temperature for 3 hand then acetonitrile (2 mL), Sodiumhydroxide (8.95 mg, 0.224 mmol) wereadded and the mixture was heated at reflux for 16 h. Product was usedfor further reaction without purification.

67. Preparation of 3-(morpholinosulfonyl)benzoic acid

3-(chlorosulfonyl)benzoic acid (250 mg, 1.133 mmol) was added to themorpholine (99 mg, 1.133 mmol) in presence of Potassiumcarbonate (313mg, 2.266 mmol) in THF (Volume: 5 ml) at room temperature and reactionmixture allowed to stirred for 12 h at room temperature. Reaction wasmonitored by TLC. After completion of the reaction, Solvent was removedby vacuum and then compound was purified by column chromatography (3%CH₃OH/CH₂Cl₂) afforded the title compound (160 mg) as a solid. ¹H NMR(400 MHz, CD₃OD): δ 8.34 (m, 1H), 8.32 (d, 1H, J=8.0 Hz), 7.99 (m, 1H),7.76 (t, 1H, J=8.0 Hz), 3.70 (m, 4H), 2.98 (m, 4H). ESI-MS: 272.0 [M+H]⁺

68. Preparation of(3-(5-chloro-2-hydroxyphenyl)-4-methyl-1H-pyrazol-1-yl)(3-(morpholinosulfonyl)phenyl)methanone

A mixture of (E)-3-(5-chloro-2-hydroxyphenyl)-2-methylacrylaldehyde (40mg, 0.203 mmol) and 4-methylbenzenesulfonohydrazide (41.7 mg, 0.224mmol) in Acetonitrile (3 mL) were stirred at room temperature for 3 hand then acetonitrile (2 mL), Sodiumhydroxide (8.95 mg, 0.224 mmol) wereadded and the mixture was heated at reflux for 16 h, thenSodiumhydroxide (12.21 mg, 0.305 mmol) and 3-(morpholinosulfonyl)benzoylchloride (88 mg, 0.305 mmol) (made from 3-(morpholinosufonyl)benzoicacid) were subsequently added and the mixture was stirred at roomtemperature for 2 h. Reaction was monitored by TLC. After completion ofthe reaction, the product was extracted with EtOAc and the organic layerwas washed with brine, dried over anhydrous Na2SO4, filtered, andsolvent was removed by vacuum. The resulting crude material was purifiedby flash column chromatography (2% CH₃OH/CH₂Cl₂) afforded the desiredproduct(3-(5-chloro-2-hydroxyphenyl)-4-methyl-1H-pyrazol-1-yl)(3-(morpholinosulfonyl)phenyl)methanone(30 mg, 0.064 mmol, 31.3% yield) as a solid. 1HNMR (400 MHz, CDCl3): δ8.28 (m, 2H), 8.20 (d, 1H, J=8.0 Hz), 7.95 (d, 1H, J=8.4 Hz), 7.70 (t,1H, J=7.6 Hz), 7.59 (d, 1H, J=2.4 Hz), 7.18 (dd, 1H, J=2.8 & 8.8 Hz),6.87 (d, 1H, J=8.4 Hz), 3.68 (m, 4H), 3.02 (m, 4H), 2.40 (s, 3H).ESI-MS: 462.0 [M+H]+

69. Preparation of 4-chloro-2-(1H-pyrazol-3-yl)phenol

A mixture of (E)-3-(5-chloro-2-hydroxyphenyl)-2-methylacrylaldehyde (40mg, 0.203 mmol) and 4-methylbenzenesulfonohydrazide (41.7 mg, 0.224mmol) in Acetonitrile (3 mL) were stirred at room temperature for 3 hand then acetonitrile (2 mL), Sodiumhydroxide (8.95 mg, 0.224 mmol) wereadded and the mixture was heated at reflux for 16 h. Product was usedfor further reaction without purification.

70. Preparation of(3-(5-chloro-2-hydroxyphenyl)-1H-pyrazol-1-yl)(3-(morpholinosulfonyl)phenyl)methanone

3-(morpholinosulfonyl)benzoic acid (50 mg, 0.184 mmol),1H-benzo[d][1,2,3]triazol-1-ol (37.4 mg, 0.276 mmol), EDC (53.0 mg,0.276 mmol) and Sodiumbicarbonate (17.03 mg, 0.203 mmol) was dissolvedin THF (10 mL) and then 4-chloro-2-(1H-pyrazol-3-yl)phenol (35.9 mg,0.184 mmol) was added at room temperature and reaction mixture wasallowed to stirred for overnight at room temperature. Reaction wasmonitored by TLC. After completion of the reaction, the solvent wasremoved by vacuum and the resulting crude material was purified by flashcolumn chromatography (2% CH₃OH/CH₂Cl₂) afforded the title compound(3-(5-chloro-2-hydroxyphenyl)-1H-pyrazol-1-yl)(3-(morpholinosulfonyl)phenyl)methanone(43 mg, 0.094 mmol, 51.0% yield) as a solid. 1HNMR (400 MHz, CDCl3): δ8.68 (s, 1H), 8.39 (d, 1H, J=7.6 Hz), 8.01 (d, 1H, J=7.6 Hz), 7.80 (d,1H, J=2.4 Hz), 7.70 (t, 1H, J=7.6 Hz), 7.53 (d, 1H, J=2.4 Hz), 7.38 (dd,1H, J=2.4 & 8.4 Hz), 7.27 (d, 1H, J=8.4 Hz), 6.52 (s, 1H), 3.75 (m, 4H),3.05 (m, 4H). ESI-MS: 448.0 [M+H]+

71. Preparation of 5-((4-methylpiperazin-1-yl)sulfonyl)nicotinohydrazide

Hydrazine (11.78 mg, 0.367 mmol) was added to the methyl5-((4-methylpiperazin-1-yl)sulfonyl)nicotinate (55 mg, 0.184 mmol) inmethanol (10 mL) and refluxed for overnight. The reaction was monitoredby TLC, after completion of the reaction, Solvent was removed by vacuumand then compound was purified by column chromatography (3%methanol/DCM) afforded the title compound5-((4-methylpiperazin-1-yl)sulfonyl)nicotinohydrazide (45 mg, 0.147mmol, 80% yield) as a solid. 1HNMR (400 MHz, CDCl3): δ 9.07 (s, 1H),9.02 (s, 1H), 8.33 (s, 1H), 7.46 (bs, 1H), 4.09 (bs, 2H), 3.05 (m, 4H),2.43 (m, 4H), 2.21 (s, 3H). ESI-MS: 300.1 [M+H]⁺

72. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-5-((4-methylpiperazin-1-yl)sulfonyl)nicotinohydrazide

1-(5-chloro-2-hydroxyphenyl)ethanone (25.07 mg, 0.147 mmol) and5-((4-methylpiperazin-1-yl)sulfonyl)nicotinohydrazide (40 mg, 0.134mmol) was dissolved in Methanol (10 ml) in the presence of acetic acidas a catalyst and then the reaction mixture was refluxed for 12 h at 70°C. Reaction was monitored by TLC. After completion of the reaction,following cooling, the solvent was removed by vacuum and the resultingcrude material was purified by flash column chromatography (2%CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-5-((4-methylpiperazin-1-yl)sulfonyl)nicotinohydrazideas a solid. 1HNMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 9.00 (s, 1H), 8.48(s, 1H), 7.41 (d, 1H, J=2.0 Hz), 7.20 (d, 1H, J=8.4 Hz), 6.90 (d, 1H,J=8.4 Hz), 3.08 (m, 4H), 2.48 (m, 4H), 2.40 (s, 3H), 2.25 (5, 3H).ESI-MS: 452.0 [M+H]+

73. Preparation of methyl 5-(morpholinosulfonyl)nicotinate

Methyl 5-(chlorosulfonyl)nicotinate (35 mg, 0.149 mmol) was added to themorpholine (25.9 mg, 0.297 mmol) in presence of Potassiumcarbonate (41.1mg, 0.297 mmol) in THF (8 mL) at room temperature and the reactionmixture was stirred for 12 h at room temperature. The reaction wasmonitored by TLC, after completion of the reaction, Solvent was removedby vacuum and then compound was purified by column chromatography (2%Methanol/DCM) afforded the product methyl5-(morpholinosulfonyl)nicotinate (26 mg, 0.090 mmol, 60.5% yield) as asolid. 1HNMR (400 MHz, CDCl3): δ 9.41 (s, 1H), 9.12 (s, 1H), 8.60 (d,1H, J=2.0 Hz), 4.01 (s, 3H), 3.76 (m, 4H), 3.07 (m, 4H).

74. Preparation of 5-(morpholinosulfonyl)nicotinohydrazide

AHydrazine (5.60 mg, 0.175 mmol) was added to the methyl5-(morpholinosulfonyl)nicotinate (25 mg, 0.087 mmol) in methanol (10 mL)and refluxed for overnight. The reaction was monitored by TLC, aftercompletion of the reaction, Solvent was removed by vacuum and thencompound was purified by column chromatography (3% methanol/DCM)afforded the title compound 5-(morpholinosulfonyl)nicotinohydrazide as asolid. 1HNMR (400 MHz, CDCl3): δ 9.15 (s, 1H), 8.98 (s, 1H), 8.41 (s,1H), 3.71 (m, 4H), 3.02 (m, 4H)

75. Preparation of(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-5-(morpholinosulfonyl)nicotinohydrazide

1-(5-chloro-2-hydroxyphenyl)ethanone (6.55 mg, 0.038 mmol) and5-(morpholinosulfonyl)nicotinohydrazide (10 mg, 0.035 mmol) wasdissolved in Methanol (3 ml) in the presence of acetic acid as acatalyst and then the reaction mixture was refluxed for 12 h at 70° C.Reaction was monitored by TLC. After completion of the reaction,following cooling, the solvent was removed by vacuum and the resultingcrude material was purified by flash column chromatography (2%CH₃OH/CH₂Cl₂) afforded the title compound(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-5-(morpholinosulfonyl)nicotinohydrazide(10 mg, 0.023 mmol, 65.2% yield) as a solid. 1HNMR (400 MHz, DMSO-d6): δ11.82 (bs, 1H), 9.35 (s, 1H), 9.08 (s, 1H), 8.52 (s, 1H), 7.66 (s, 1H),7.34 (d, 1H, J=8.4 Hz), 6.94 (d, 1H, J=8.8 Hz), 3.63 (m, 4H), 2.99 (m,4H), 2.50 (s, 3H). ESI-MS: 439.1 [M+H]+

76. General Biochemical and Cell Materials and Methods

LSD1 activity was determined using a LSD1 Inhibitor Screening Assay Kit(Cayman Chemical Item Number 700120) purchased from Cayman ChemicalCompany (Ann Arbor, Mich.). Recombinant (expressed in baculovirusinfected BTI insect cells) monoamine oxidase A and monoamine oxidase B(Catalog No. M7316 and M7441, respectively) were purchased fromSigma-Aldrich Co. LLC. (St. Louis, Mo.). MAO-Glo™ Assay Kit waspurchased from Promega Corporation (Madison, Wis.). ATPlite™Luminescence Assay System (e.g. Catalog No. V1401) was purchased fromPerkin Elmer Inc. (Waltham, Massachussetts).

77. Cell Culture

Cancer cell lines were obtained from ATCC. Cells were cultured accordingto the procedures provided. Cell-lines used included those shown inTable 4 below. In addition to the supplements indicated in Table 4,media were also supplemented with 1% penicillin/streptomycin (100 IU/mLpenicillin and 100 μg/mL streptomycin). Cells were cultured at 37° C.and 5% CO₂. ATCC is the American Type Culture Collection (Manassas,Va.).

TABLE 4 ATCC ® Cell-line Number Organ/tissue/pathology* Culture MediaAN3 CA HTB-111 ™ Uterine/endometrium/ Eagle's Minimum Essentialadenocarcinoma Medium supplemented with 10% FCS** BT-20 HTB-19 ™Breast/carcinoma Eagle's Minimum Essential Medium supplemented with 10%FCS BT-549 HTB-122 ™ Breast/ductal carcinoma RPMI-1640 Mediumsupplemented with 0.023 IU/ml insulin and 10% FCS HCT 116 CCL-247 ™Colon/colorectal carcinoma McCoy's 5a Medium Modified supplemented with10% FCS HER218*** Not applicable Breast/adenocarcinoma RPM1-1640 Mediumsupplemented and 10% FCS MCF7 HTB-22 ™ Breast/adenocarcinoma Eagle'sMinimum Essential Medium supplemented with 0.01 mg/ml bovine insulin and10% FCS. MDA-MB-231 HTB-26 ™ Breast/adenocarcinoma Leibovitz's L-15Medium supplemented with 10% FCS MDA-MB-435S HTB-129 ™ Pleural effusion;likely Leibovitz's L-15 Medium melanoma supplemented with 0.01 mg/mlbovine insulin, •0.01 mg/ml glutathione, and 10% FCS MDA-MB-468HTB-132 ™ Breast/adenocarcinoma Leibovitz's L-15 Medium supplementedwith 10% FCS PANC-1 CRL-1469 ™ Pancreas/duct/epithelioid Dulbecco'sModified Eagle's carcinoma Medium supplemented with 10% FCS PC-3CRL-1435 ™ Prostate adenocarcinoma F-12K Medium supplemented with 10%FCS SK-N-MC HTB-10 ™ Brain/neuroepithelioma Eagle's Minimum EssentialMedium supplemented with 10% FCS T-47D HTB-133 ™ Breast/ductal carcinomaRPMI-1640 Medium supplemented with 0.2 units/ml bovine insulin and 10%FCS U-87 MG HTB-14  ™ Brain/glioblastoma, Eagle's Minimum Essentialastrocytoma Medium supplemented with 10% FCS *All organ/tissue sourceswere of human origin. **FCS is Fetal Calf Serum ***MCF7 cell-linederivative characterized by non-nuclear estrogen receptor and highlevels of HER2 (Massarweh S, et al. (2008) Cancer Research 68: 826-33).

78. LSD1 Histone Demethylase Assay

The primary assay for compound inhibitory activity was the LSD1Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor,Mich.; Cayman Chemical Item Number 700120). Briefly, test compounds werediluted to 20× the desired test concentration in 100% DMSO and 2.5 μL ofthe diluted drug sample was added to a black 384-well plate. The LSD1enzyme stock was diluted 17-fold with assay buffer and 40 μM of thediluted LSD1 enzyme was added to the appropriate wells. The reactionmixture comprised horseradish peroxidase, dimethyl K4 peptide(corresponding to the first 21 amino acids of the N-terminal tail ofhistone H3), and 10-acetyl-3,7-dihydroxyphenoxazine was then added towells. Generation of resorufin (generated by reacting with H₂O₂ producedin the reaction) was analyzed on an Envision microplate reader with anexcitation wavelength of 530 nm and an emission wavelength of 595 nm.

79. Monoamine Oxidase (“MAO”) Assay

Inhibition of monoamine oxidase activity was carried used using theMAO-Glo™ Assay Kit according to the manufacturer's suggested protocol.Briefly, 6.25 μL of test compound was added to each well of a 384-wellplate. Enzyme (either MAO A or B) was added (12.5 μL in 2× buffercontaining 1 μg protein) and allowed to incubate for 5 minutes. Finally,6.25 μL of 4×MAO substrate was added to each well. Following a one hourincubation, 25 μL of Luciferin detection reagent was added to each well,and incubated for 20 minutes. Luminescence was then measured on anEnvision microplate reader. Representative data used to determine IC₅₀for inhibition of each MAO isoform is provided in FIG. 4, andrepresentative data for several compounds is summarized in Table 8below.

80. Cell Viability Assay

Cell viability was determined using ATPlite™ Luminescence Assay System(PerkinElmer Inc., Waltham, Massachussetts) using the various cell-linesdescribed above and in Table 4. Briefly, cells were seeded in 96-wellplates and then treated with different concentrations of inhibitor (0.1%final DMSO concentration). After 96-hours of incubation, ATPlitedetection reagent was added directly to the culture well. Luminescencewas read 5 minutes later on an Envision microplate reader.Representative IC₅₀ data for inhibition of cell growth with variouscell-lines is provided below in Tables 6, 7, and 9.

81. Real-Time PCR

Briefly, T-47D cells were seeded in 96-well plates and treated withconcentrations of inhibitors as indicated. Cell lysates, Reversetranscription, and single color syber green realtime PCR was performedusing the Cells-to-Ct kit (Life Technologies). Transcript levels of hemeoxygenase (HMOX) were normalized to hypoxanthinephosphoribosyltransferase (HPRT) and β-actin. The primers used inreal-time PCR are shown below in Table 5, and representative data forthe effect of disclosed compounds on HMOX expression are provided inTables 6 and 7.

TABLE 5 Primer Designation Amplification Target Sequence HMOX_F Hemeoxygenase AACTTTCAGAAGGGCCAGGT HMOX_R Heme oxygenaseGTAGACAGGGGCGAAGACTG HPRT_F Hypoxanthine TGCTGAGGATTTGGAAAGGGTGphosphoribosyltransferase HPRT_R Hypoxanthine CCTTGAGCACACAGAGGGCTACphosphoribosyltransferase B-Actin_F β-actin CTGGAACGGTGAAGGTGACAB-Actin_R β-actin AAGGGACTTCCTGTAACAACGCA

82. IC₅₀ Calculation

IC₅₀ values are determined using GraphPad Prism 5 software. The datawere entered as an X-Y plot into the software as percent inhibition foreach concentration of the drug. The concentration values of the drugwere log transformed and the nonlinear regression was carried out usingthe “sigmoidal dose-response (variable slope)” option within theGraphPad software to model the data and calculate IC₅₀ values. The IC₅₀values reported are the concentration of drug at which 50% inhibitionwas reached.

83. Compound Activity

The ability of representative disclosed compounds to modulate variousbiochemical and cellular activities was determined using the assaysdescribed above. The results are shown in the tables below. The IC₅₀(μM) for inhibition of either LSD1 activity or cell growth using T-47Dcells is shown in Tables 6 and 7. In addition, the effect ofrepresentative compounds on heme oxygenase (HMOX) expression is alsoshown in Tables 6 and 7. The IC₅₀ for inhibition of monoamine oxidases A(“MAO A”) and B (“MAO B”) by representative compounds compared to acontrol compound, tranylcypromine, is shown in Table 8. The effect ofCompound No. 12 (in reference to the compound number used in Table 7, or(E)-N′-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide)on cell growth for various cell-lines in shown in Table 9. If an IC₅₀ orother assay result is indicated as “n.d.”, it was not determined in theindicated assay.

Compound 12 was used to evaluate sensitivity in a panel of cancer celllines (Table 9). Cell line sensitivity to compound 12 in this viabilityassay varied by one log, with IC₅₀ values around 300 nM to just under 3μM. For comparison among the representative compounds, IC₅₀ values weredetermined in T-47D cells (see Tables 6 and 7). With few exceptions, itwas observed that T-47D cells were sensitive to test compounds that wereactive in the LSD1 biochemical assay, and were less sensitive to thecompounds which showed less activity in the LSD1 assay.

In order to add an additional level of analysis of LSD1 inhibition incell culture by these compounds, expression array experiments wereperformed to evaluate transcriptional changes induced by compound 12(data not shown). These data indicated that heme oxygenase 1 (HMOX1) wasone of the most consistently up-regulated genes across multiple celllines following treatment with this compound. As HMOX1 is known to beregulated by H3 methylation in the promoter (Krieg, A. J., et al. MolCell Biol 2010, 30 (1), 344-53), the effect of the test compounds onHMOX1 expression in T-47D cells was determined (see Tables 6 and 7). Thedata show that the representative compounds which are associated withupregulation of HMOX1 expression are also associated inhibitory activityin the LSD1 assay and the cell viability assay.

LSD1 has a high structural homology of to the monoamine oxidase familyof enzymes (17.6% for both monoamine oxidase A and B; MAO A and B,respectively; e.g. see Gooden, D. M., et al. Bioorg Med Chem Lett 2008,18 (10), 3047-51). Selective activity of the representative compoundsfor LSD1 compared to either MAO A or MAO B, is a desirable property fortherapeutic compounds targeting LSD1. The specificity of compound 1 andcompound 12 were tested in MAO biochemical assays described herein (seeFIG. 3 for representative results which are summarized in Table 8). Inthis assay, the known MAO inhibitor tranylcypromine exhibited activityagainst both MAO A and B. In contrast, compound 1 exhibited comparableactivity to tranylcypromine against MAO B, but showed no activityagainst MAO A. However, compound 12 does not exhibit activity againsteither MAO enzyme (>300 μM). Compounds 18 and 24 were also tested, andexhibited no activity against MAO A or B, and the results are providedin Table 8. These results demonstrate that the representative compoundshave specificity for LSD1 with significantly reduced effect on the MAOenzymes. It should be noted that both MAO A and B differ from LSD1 inthat the FAD is covalently bound to the enzyme through a thioetherlinkage with Cys406 and Cys397, respectively (Kearney, E. B., et al.European Journal of Biochemistry 1971, (24), 321-327; and Bach, A. W.,et al. Proc Natl Acad Sci USA 1988, (85), 4934-4938).

TABLE 6 HMOX LSD1 Cell expression Activity, Growth, (fold- No. StructureIC₅₀ (μM) IC₅₀ (μM) induction)  1

0.218 2.7 2.3  2

0.275 0.821 13  3

0.291 0.971 15.1  4

0.196 0.096 20.3  5

0.333 0.615 31.5  6

>3 >10 1.9  7

>3 >10 1.1  8

>3 >10 0.9  9

0.013 0.524 31.7 10

>10 >10 1.0

TABLE 7 HMOX LSD1 Cell expression Activity, Growth, (fold- No. StructureIC₅₀ (μM) IC₅₀ (μM) induction) 11

0.128 0.352 31.3 12

0.013 0.649 26.9 13

>3   >10    ND 14

>3   >10     1.1 15

>3   >10    ND 16

>3   >10     0.9 17

>3   1.700 ND 18

0.013 0.565 56.4 19

>3   1.375 ND 20

>3   0.270 ND 21

>3   0.616 ND 22

>3   ND ND 23

0.519 ND ND 24

0.028 ND ND 25

0.049 ND 50.3 26

 0.0095 ND ND 27

>3   ND ND 28

>3   ND ND 29

 0.0087 ND ND 30

ND ND ND 31

ND ND ND 32

>3   ND ND 33

ND ND ND 34

ND ND ND 35

<0.01   ND ND 36

ND ND ND 37

>3   ND ND 38

>3   ND ND 39

ND ND ND 40

ND ND ND

TABLE 8 MAO A, MAO B, No. Structure IC50 (μM) IC50 (μM) —

2.1 3.6  1

88.5 1.3 12

>300 >300 18

>300 >300 24

>300 >300

TABLE 9

Cell-line Cell Growth, IC50 (μM) AN3 Ca 0.356 BT-20 0.489 BT-549 1.010HCT 116 0.614 HER218 0.612 Hs-578-T 1.700 HT29 0.429 MCF-7 0.637MDA-MB-231 1.040 MDA-MB-235 0.728 MDA-MB-435 1.440 MDA-MB-468 2.730 MIAPaCa-2 0.468 PANC-1 1.104 PC-3 2.160 SK-N-MC 0.329 T-47D 0.649 U87 1.160

84. Prophetic In Vivo Anti-Tumor Effects: Cell-Line Xenograft Model

The following examples of the in vivo effect of the disclosed compoundsare prophetic. Generally agents which modulate the regulation ofchromatin, including histone demethylase inhibitors, display efficacy inpreclinical models of cancer. In vivo effects of the compounds describedin the preceding examples are expected to be shown in various animalmodels of cancer known to the skilled person, such as tumor xenograftmodels. These models are typically conducted in rodent, most often inmouse, but may be conducted in other animal species as is convenient tothe study goals. Compounds, products, and compositions disclosed hereinare expected to show in vivo effects in various animal models of cancerknown to the skilled person, such as mouse tumor xenograft models.

In vivo effects of compounds can be assessed with in a mouse tumorxenograft study, one possible study protocol is described herein.Briefly, cells (2 to 5×10⁶ in 100 mL culture media) were implantedsubcutaneously, e.g. by subcutaneous injection, in the right hind flankof athymic nu/nu nude mice (5 to 6 weeks old, 18-22 g). For testcompounds of the present invention, a typical cell-line used for thetumor xenograft study would be AN3 CA or BT-20. Other suitablecell-lines for these studies are BT-549, HCT 116, HER218, MCF7,MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468, PANC-1, PC-3, SK-N-MC,T-47D, and U-87 MG cells. The cells are cultured prior to harvesting forthis protocol as described herein.

Following implantation, the tumors are allowed to grow to about 100 mm³,typically about 6-18 days post-implantation, before the animals arerandomized into treatment groups (e.g. vehicle, positive control andvarious dose levels of the test compound); the number of animals pergroup is typically 8-12. Day 1 of study corresponds to the day that theanimals receive their first dose. The efficacy of a test compound can bedetermined in studies of various lengths dependent upon the goals of thestudy. Typical study periods are for 14, 21 and 28-days. The dosingfrequency (e.g. whether animals are dosed with test compound daily,every other day, every third day or other frequencies) is determined foreach study depending upon the toxicity and potency of the test compound.A typical study design would involve dosing daily (M-F) with the testcompound with recovery on the weekend. Throughout the study, tumorvolumes and body weights are measured twice a week. At the end of thestudy the animals are euthanized and the tumors harvested and frozen forfurther analysis. Alternatively, tumors may be processed immediately foranalysis, e.g. fixed in buffered-formalin, paraffin embedded, andsectioned for hematoxylin/eosin staining and further immunohistochemicalanalysis for desired oncology markers.

For example, compounds of the invention, or a pharmaceuticallyacceptable salt, solvate, polymorph, hydrate and the stereochemicallyisomeric form thereof, are expected to show such in vivo effects.

85. Prophetic In Vivo Anti-Tumor Effects: Tumor Graft Model

Alternatively, it can be desirable to assess the in vivo efficacy of thedisclosed compounds in a tumor explant or tumor graft animal models(e.g. see Rubio-Viqueira B., et al. Clin Cancer Res. (2006)12:4652-4661; Fiebig, H. H., Maier, A. and Burger, A. M. Eur. J. Canc.(2004) 40:802-820; and DeRose, Y. S., et al. “Patient-derived tumorgrafts authentically reflect tumor pathology, growth, metastasis anddisease outcomes.” (2011) Nat. Med., in press). These models can providehigher quality information on in vivo effects of therapeutic compounds.It is believed tumor graft models are more authentic in vivo models ofmany types of cancer, e.g. human breast cancer, with which to examinethe biology of tumors and how they metastasize. Engraftment of actualpatient tumor tissues into immunodeficient mice (termed ‘tumor grafts’)provides improvement over implantation of cell lines, in terms ofphenocopying human tumors and predicting drug responses in patients(Clarke, R. Breast Cancer Res (2009) 11 Suppl 3, S22; Press, J. Z., etal. Gynecol Oncol (2008) 110:56-264; Kim, M. P., et al. Nat Protoc(2009) 4:670-1680; Daniel, V. C., et al. Cancer Res (2009) 69:3364-3373;and Ding, L., et al. Nature (2010) 464:999-1005).

Briefly, tissue samples will be collected from informed, consentedpatients at Huntsman Cancer Hospital/University of Utah under anapproved IRB protocol. Samples will be collected and de□identified bythe Huntsman Cancer Institute Tissue Resource and Application Corefacility before being obtained for implantation. It is anticipated thatall primary tumors will be from individuals that had not receivedchemotherapy prior to tissue collection, and all metastatic effusionswill be from individuals that had been treated with chemotherapy,hormone therapy, and/or radiation therapy. The University of UtahInstitutional Animal Care and Use Committee will review and approve allmouse experiments. It is anticipated that a minimum of three mice perexperimental group will be used, and only female mice will be used forstudies involving breast cancer tumors. A single fragment of fresh orfrozen tumor (˜8 mm3), or about 10⁶ cells in matrigel, is implanted intocleared inguinal mammary fat pads of 3-4 week old female NOD/SCID mice.At the same, interscapular estrogen pellets are subcutaneously implantedin mice with ER+ tumors. Tumor growth is measured weekly using calipers.When tumors reach about 150-2,000 mm³, the mice are euthanized, andtissue fragments are re-transplanted into another cohort of mice, frozenfor later use, and/or analyzed for histology, gene expression, and DNAcopy number. Tumor volumes are calculated using the formula0.5×length×(width)². For experiments to determine estrogen dependence,ER⁺ tumors are implanted into mice as described above, in the presenceor absence of intrascapular estrogen pellets and with or without aconcurrent surgical procedure to remove the ovaries, which is performedaccording to standard methods.

Freshly harvested tumor tissues from patients or mice are cut into ˜8mm3 pieces and stored in liquid nitrogen, in a solution of 95% FBS and5% DMSO for later implantation. Alternatively, the tissue is digestedwith collagenase solution (1 mg/ml collagenase [Type IV, Sigma] in RPMI1640 supplemented with 2.5% FBS, 10 mM HEPES, 10 μg/mLpenicillin-streptomycin) at 37° C. for 40-60 min, while shaking at 250rpm. Digested tissue is strained to remove debris and washed in humanbreast epithelial cell (HBEC) medium (DMEM F/12 supplemented with 10 mMHEPES, 5% FBS, 1 mg/mL BSA, 0.5 μg/mL hydrocortisone, 50 μg mLGentamycin, 1 μg/mL ITS-X100) three times. The pellet is resuspended infreezing medium (5% FBS and 10% DMSO in HBEC medium) and stored inliquid nitrogen.

To assess the effect of a disclosed compound, tumors in mice are allowedto grow to about 100 mm³, typically about 6-18 days post-implantation,before the animals are randomized into treatment groups (e.g. vehicle,positive control and various dose levels of the test compound); thenumber of animals per group is typically 8-12. Day 1 of studycorresponds to the day that the animals receive their first dose. Theefficacy of a test compound can be determined in studies of variouslengths dependent upon the goals of the study. Typical study periods arefor 14, 21 and 28-days. The dosing frequency (e.g. whether animals aredosed with test compound daily, every other day, every third day orother frequencies) is determined for each study depending upon thetoxicity and potency of the test compound. A typical study design wouldinvolve dosing daily (M-F) with the test compound with recovery on theweekend. Throughout the study, tumor volumes and body weights aremeasured twice a week. At the end of the study the animals areeuthanized and the tumors harvested and frozen for further analysis.Alternatively, tumors may be processed immediately for analysis, e.g.fixed in buffered-formalin, paraffin embedded, and sectioned forhematoxylin/eosin staining and further immunohistochemical analysis fordesired oncology markers.

For example, compounds of the invention, or a pharmaceuticallyacceptable salt, solvate, polymorph, hydrate and the stereochemicallyisomeric form thereof, are expected to show such in vivo effects.

86. Prophetic Pharmaceutical Composition Examples

“Active ingredient” as used throughout these examples relates to one ormore of the compounds of the invention, or a pharmaceutically acceptablesalt, solvate, polymorph, hydrate and the stereochemically isomeric formthereof. The following examples of the formulation of the compounds ofthe present invention in tablets, suspension, injectables and ointmentsare prophetic.

Typical examples of recipes for the formulation of the invention are asgiven below. Various other dosage forms can be applied herein such as afilled gelatin capsule, liquid emulsion/suspension, ointments,suppositories or chewable tablet form employing the disclosed compoundsin desired dosage amounts in accordance with the present invention.Various conventional techniques for preparing suitable dosage forms canbe used to prepare the prophetic pharmaceutical compositions, such asthose disclosed herein and in standard reference texts, for example theBritish and US Pharmacopoeias, Remington's Pharmaceutical Sciences (MackPublishing Co.) and Martindale The Extra Pharmacopoeia (London ThePharmaceutical Press).

The disclosure of this reference is hereby incorporated herein byreference.

a. Pharmaceutical Composition for Oral Administration

A tablet can be prepared as follows:

Component Amount Active ingredient 10 to 500 mg Lactose 100 mgCrystalline cellulose  60 mg Magnesium stearate 5 Starch (e.g. potatostarch) Amount necessary to yield total weight indicated below Total(per capsule) 1000 mg 

Alternatively, about 100 mg of a disclosed compound, 50 mg of lactose(monohydrate), 50 mg of maize starch (native), 10 mg ofpolyvinylpyrrolidone (PVP 25) (e.g. from BASF, Ludwigshafen, Germany)and 2 mg of magnesium stearate are used per tablet. The mixture ofactive component, lactose and starch is granulated with a 5% solution(m/m) of the PVP in water. After drying, the granules are mixed withmagnesium stearate for 5 min. This mixture is moulded using a customarytablet press (e.g. tablet format: diameter 8 mm, curvature radius 12mm). The moulding force applied is typically about 15 kN.

Alternatively, a disclosed compound can be administered in a suspensionformulated for oral use. For example, about 100-5000 mg of the desireddisclosed compound, 1000 mg of ethanol (96%), 400 mg of xanthan gum, and99 g of water are combined with stirring. A single dose of about 10-500mg of the desired disclosed compound according can be provided by 10 mlof oral suspension.

In these Examples, active ingredient can be replaced with the sameamount of any of the compounds according to the present invention, inparticular by the same amount of any of the exemplified compounds. Insome circumstances it may be desirable to use a capsule, e.g. a filledgelatin capsule, instead of a tablet form. The choice of tablet orcapsule will depend, in part, upon physicochemical characteristics ofthe particular disclosed compound used.

Examples of alternative useful carriers for making oral preparations arelactose, sucrose, starch, talc, magnesium stearate, crystallinecellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, carboxymethyl cellulose, glycerin, sodiumalginate, gum arabic, etc. These alternative carriers can be substitutedfor those given above as required for desired dissolution, absorption,and manufacturing characteristics.

The amount of a disclosed compound per tablet for use in apharmaceutical composition for human use is determined from bothtoxicological and pharmacokinetic data obtained in suitable animalmodels, e.g. rat and at least one non-rodent species, and adjusted basedupon human clinical trial data. For example, it could be appropriatethat a disclosed compound is present at a level of about 10 to 1000 mgper tablet dosage unit.

b. Pharmaceutical Composition for Injectable Use

A parenteral composition can be prepared as follows:

Component Amount Active ingredient 10 to 500 mg Sodium carbonate 560 mg*Sodium hydroxide  80 mg* Distilled, sterile water Quantity sufficient toprepare total volume indicated below. Total (per capsule) 10 ml perampule *Amount adjusted as required to maintain physiological pH in thecontext of the amount of active ingredient, and form of activeingredient, e.g. a particular salt form of the active ingredient.

Alternatively, a pharmaceutical composition for intravenous injectioncan be used, with composition comprising about 100-5000 mg of adisclosed compound, 15 g polyethylenglycol 400 and 250 g water in salinewith optionally up to about 15% Cremophor EL, and optionally up to 15%ethyl alcohol, and optionally up to 2 equivalents of a pharmaceuticallysuitable acid such as citric acid or hydrochloric acid are used. Thepreparation of such an injectable composition can be accomplished asfollows: The disclosed compound and the polyethylenglycol 400 aredissolved in the water with stirring. The solution is sterile filtered(pore size 0.22 μm) and filled into heat sterilized infusion bottlesunder aseptic conditions. The infusion bottles are sealed with rubberseals.

In a further example, a pharmaceutical composition for intravenousinjection can be used, with composition comprising about 10-500 mg of adisclosed compound, standard saline solution, optionally with up to 15%by weight of Cremophor EL, and optionally up to 15% by weight of ethylalcohol, and optionally up to 2 equivalents of a pharmaceuticallysuitable acid such as citric acid or hydrochloric acid. Preparation canbe accomplished as follows: a desired disclosed compound is dissolved inthe saline solution with stirring. Optionally Cremophor EL, ethylalcohol or acid are added. The solution is sterile filtered (pore size0.22 μm) and filled into heat sterilized infusion bottles under asepticconditions. The infusion bottles are sealed with rubber seals.

In this Example, active ingredient can be replaced with the same amountof any of the compounds according to the present invention, inparticular by the same amount of any of the exemplified compounds.

The amount of a disclosed compound per ampule for use in apharmaceutical composition for human use is determined from bothtoxicological and pharmacokinetic data obtained in suitable animalmodels, e.g. rat and at least one non-rodent species, and adjusted basedupon human clinical trial data. For example, it could be appropriatethat a disclosed compound is present at a level of about 10 to 1000 mgper tablet dosage unit.

Carriers suitable for parenteral preparations are, for example, water,physiological saline solution, etc. which can be used withtris(hydroxymethyl)aminomethane, sodium carbonate, sodium hydroxide orthe like serving as a solubilizer or pH adjusting agent. The parenteralpreparations contain preferably 50 to 1000 mg of a disclosed compoundper dosage unit.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A compound having a structure represented by aformula (I)

or a formula (II)

wherein m is 0 or 1; n is an integer from 0 to 3; X is selected from thegroup consisting of OH, NO₂ and F; Z is selected from the groupconsisting of N and CH; R₁ is selected from the group consisting ofhalo, C1-C3 haloalkyl, and C1-C3 polyhaloalkyl; each of R₂, R₃, and R₄is independently selected from the group consisting of hydrogen, halo,hydroxyl, cyano, amino, C2-C6 alkalkoxy, C1-C6 alkoxy, C1-C6 alkyl,C1-C6 polyhaloalkyl, and C1-C6 haloalkyl; R₅ is selected from the groupconsisting of NR₆ R₇, C1-C6 alkyl, C3-C6 cycloalkyl,

and Cy, and substituted with 0-3 groups independently selected fromhalo, hydroxyl, amino, C2-C6 alkalkoxy, C1-C6 alkylalcohol, C1-C6alkoxy, C1-C6 alkyl, C1-C6 polyhaloalkyl, C1-C6 haloalkyl, C3-C6cycloalkyl, and Cy; Cy is a heterocycloalkyl selected from the groupconsisting of aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl,azepanyl, oxazolidinyl, imidazolidinyl, pyrazolidinyl, piperazinyl,oxazinanyl, morpholinyl, hexahydrophyrimidinyl, andhexahydropyridazinyl; and each of R₆ and R₇ is independently selectedfrom the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl,and C3-C6 heterocycloalkyl; or a pharmaceutically acceptable saltthereof.
 2. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 3. A method for the treatment of a disorder ofuncontrolled cellular proliferation in a mammal, the method comprisingthe step of administering to the mammal an effective amount of acompound of claim
 1. 4. A method for decreasing histone demethylaseactivity in a mammal, the method comprising the step of administering tothe mammal an effective amount of a compound of claim
 1. 5. A compoundselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 6. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof claim 5 and a pharmaceutically acceptable carrier.
 7. A method forthe treatment of a disorder of uncontrolled cellular proliferation in amammal, the method comprising the step of administering to the mammal aneffective amount of a compound of claim
 5. 8. A method for decreasinghistone demethylase activity in a mammal, the method comprising the stepof administering to the mammal an effective amount of a compound ofclaim
 5. 9. A compound having a structure represented by a formula:

or a pharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof claim 9 and a pharmaceutically acceptable carrier.
 11. A method forthe treatment of a disorder of uncontrolled cellular proliferation in amammal, the method comprising the step of administering to the mammal aneffective amount of a compound of claim
 9. 12. A method for decreasinghistone demethylase activity in a mammal, the method comprising the stepof administering to the mammal an effective amount of a compound ofclaim
 9. 13. A compound having a structure represented by a formula:

or a pharmaceutically acceptable salt thereof.
 14. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof claim 13 and a pharmaceutically acceptable carrier.
 15. A method forthe treatment of a disorder of uncontrolled cellular proliferation in amammal, the method comprising the step of administering to the mammal aneffective amount of a compound of claim
 13. 16. A method for decreasinghistone demethylase activity in a mammal, the method comprising the stepof administering to the mammal an effective amount of a compound ofclaim 13.